A fundamental step during cardiac remodeling is the transdifferentiation of quiescent cardiac fibroblasts into cardiac myofibroblasts, which are characterized by their enhanced capacity to express and secrete extracellular matrix components and regulators. Consequently, an adaptation of the endoplasmic reticulum (ER) is necessary to cope with the increased cargo load. We hypothesized that interference with this adaptation could result in unresolvable ER stress and thus in the degradation of ER-targeted mRNAs by the endonuclease IRE1alpha and a translational repression by the PERK pathway. To test our hypothesis, we treated primary human cardiac myofibroblasts (MyoF) in 2D cultures as well as human foreskin fibroblasts with efficient ER stress inducers, like the sarco/endoplasmic reticulum calcium ATPase inhibitor thapsigargin (TG). This resulted in a strong induction of ER stress for both cell types, as shown by the increased expression of the major ER chaperone BiP (encoded by HSPA5) and the pro-apoptotic transcription factor CHOP (encoded by DDIT3). We next investigated the effect of TG on the expression of ECM components and found a strong decline in pro-collagen 1 in both cell types. As TG results in a depletion of the ER calcium, we analyzed if inhibition of the store-operated calcium entry by BTP2, which moderately reduced the ER calcium levels, could have a similar effect in MyoF. In line with our hypothesis, we found that BTP2 increased the transcript levels of DDIT3, indicating ER stress, and reduced the mRNA levels as well as the protein levels of collagen 1. To test if the PERK pathway, and thus the translational repression, is involved in the observed decline in pro-collagen 1 expression, MyoF were treated with a combination of TG and the PERK inhibitor GSK2656157, which resulted in a partial restoration of pro-collagen 1 expression. Surprisingly, PERK inhibition alone also induced moderate ER stress, demonstrated by an increase in DDIT3 and a decline in COL1A1. However, in this condition no increase in HSPA5 could be detected. Finally, we could show that the ER stress inducers BTP2 and GSK2656157 reduced the expression of COL1A1 in human engineered connective tissues, which resulted in a decline in tissue stiffness. In conclusion, induction of ER stress in cardiac myofibroblasts blocks the production of collagen and thus could serve as an anti-fibrotic therapeutic strategy.