The construction of the cathedral was entrusted to the Parler's building workshop in 1388. Johann, the son of Peter Parler, took charge of the whole project. The church was initially designed as a triple-nave cathedral-type building, with an ambulatory around the chancel. The construction started from the east. In the beginning, the progress was very fast - in 1391, the chancel with unfinished vaulting was consecrated, and in 1403, baptising and burying was permitted by a papal bull. In the same year, the cathedral got its name: New Corpus Christi and St Barbara's Chapel. A year later, Johann Parler changed the plans when he decided to add one more aisle to each side of the nave-aisles. The result was a cathedral with five naves. Before the outbreak of the Hussite wars, the cathedral was built to the height of the ambulatory level in its present length, and only the ambulatory chapels and the outer nave-aisle were vaulted; the choir and the central nave were covered by a temporary ceiling at the ambulatory level.

The Hussite wars stopped the construction. The works resumed in 1481 thanks to the Royal Hofmeister, Michal Smíšek of Vrchoviště, who organised a collection for the church vaulting. Master Hanuš, who was the stonemason of the Parlers' school, led the works; the construction continued according to the Johann Parler's plan. Master Hanuš worked on the cathedral until he died in 1489. In a relatively short time, he managed to vault the endings of the chapels as well as the nave-aisles and to partly build a triforium (a wall-thick ambulatory under the choir windows).

While the cathedral gained a unique exterior decoration during the Rejsek's era, in the next period, associated with the master architect Benedikt Ried, the concept of the five-aisle cathedral changed radically. Ried approached the construction quite originally: he attached the inner galleries to the upper part of the cathedral and thus created a second church upstairs consisting of three naves above the ground five naves. By doing that he fulfilled the need of free space for the preacher's speech and choral singing and achieved an exceptional brightness of the interior. In 1532, while the master Benedikt was still alive, the cathedral was roofed by three typical tent-like roofs.

Benedikt Ried worked on the St Barbara's Cathedral until 1534. Before his death, he drew up further plans based on which Mikuláš and Jan Vlach continued the work since 1540. They used a stellar vault with intertwined curved ribs to roof the triple-nave hall, built the organ loft and finished the tracery of the windows. The works went on slowly till 1558 when the cathedral was provisionally closed by a temporary wall in the west due to lack of funds. In 1595, this wall was extended with a gable and decorated with a two-cubit high chalice.

In the period following the Battle of White Mountain, when the recatholicisation was taking place in the Czech lands, the Jesuits came to the utraquist Kutná Hora (1626). They took over the administration of the cathedral. In many ways, they adapted it to the needs of their Order and to the Baroque style. The Gothic frescoes were painted over with white paint. The chalice was taken down, and the original tented roof was replaced by a gabled one with three ridge turrets. The late Gothic altar was replaced by a Baroque one and pews for the faithful were placed in the cathedral. In the years 1667 - 1670, the Jesuits built a Jesuit college next to the cathedral, which in 1727 they connected to the cathedral by an elevated roofed gallery made of stone and leading to the sacristy. This passageway was demolished at the end of the 18th century when the building served as military quarters.

In 1773, when the Jesuits Order was abolished, the cathedral was passed to the State religious fund. The fund did not take good care of the cathedral, so it gradually fell into disrepair until 1877, when the Museum and archaeological society Vocel was founded. This society resolved to restore the cathedral into its original shape. The reconstruction was led by architect Josef Mocker and later by Ludvík Lábler. The temporary western wall was demolished, and a new façade was built with an extra vaulted bay. The late Gothic frescoes were restored, the Baroque main altar was replaced by a neogothic copy of the original altar. In 1905, the cathedral was consecrated again and was handed over to the Archdeanery Kutná Hora for administration.