Calcium (Ca2+) is a fundamental regulator of cell signaling and function. Thapsigargin (Tg) blocks the sarco/endoplasmic reticulum (ER) Ca2+-ATPase (SERCA), disrupts Ca2+ homeostasis, and causes cell death. However, the exact mechanisms whereby SERCA-inhibition induces cell death are incompletely understood. Here, we report that low (0.1 μM) concentrations of Tg and Tg analogs with various long-chain substitutions at the O(8) position extensively inhibit SERCA1a-mediated Ca2+ transport. We also found that in both prostate and breast cancer cells, exposure to Tg or Tg analogs for 1 day caused extensive drainage of the ER Ca2+ stores. This Ca2+ depletion was followed by markedly reduced cell proliferation rates and morphological changes that developed over 2–4 days and culminated in cell death. Interestingly, these changes were not accompanied by bulk increases in cytosolic Ca2+ levels. Moreover, knockdown of two key store-operated Ca2+ entry (SOCE) components, Orai1 and STIM1, did not reduce Tg cytotoxicity, indicating that SOCE and Ca2+ entry are not critical for Tg-induced cell death. However, we observed a correlation between the abilities of Tg and Tg analogs to deplete ER Ca2+ stores and their detrimental effects on cell viability. Furthermore, caspase activation and cell death were associated with a sustained unfolded protein response (UPR). We conclude that ER Ca2+ drainage and sustained UPR activation are key for initiation of apoptosis at low concentrations of Tg and Tg analogs, whereas high cytosolic Ca2+ levels and SOCE are not required.