Eeyarestatin 1 (ES1) inhibits p97-dependent protein degradation, Sec61-dependent protein translocation into the endoplasmic reticulum (ER) and vesicular transport within the endomembrane system. Here, we show that ES1 impairs Ca2+ homeostasis by enhancing the Ca2+ leakage from mammalian ER. A comparison of various ES1 analogues suggested that the 5-nitrofuran (5-NF) ring of ES1 is crucial for this effect. Accordingly, the analogue ES24, which conserves the 5-NF domain of ES1, selectively inhibited protein translocation into the ER, displayed the highest potency on ER Ca2+ leakage of ES1 analogues studied and induced Ca2+-dependent cell death. Using siRNA mediated knockdown of Sec61α, we identified Sec61 complexes as the targets that mediate the gain of Ca2+ leakage induced by ES1 and ES24. By interacting with the lateral gate of Sec61α, ES1 and ES24 likely capture Sec61 complexes in a Ca2+-permeable, open state, in which Sec61 complexes allow Ca2+ leakage but are translocation-incompetent.