The Golgi/secretory pathway Ca2+/Mn2+ transport ATPase (SPCA1a) is implicated in breast cancer and Hailey-Hailey disease. Here, we purified recombinant human SPCA1a from Saccharomyces cerevisiae and measured Ca2+ dependent ATPase activity following reconstitution in proteoliposomes. The purified SPCA1a displays a higher apparent Ca2+ affinity and lower maximal turnover rate than the purified sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA1a). The lipids cholesteryl hemisuccinate, linole-/oleamide and phosphatidyl ethanolamine inhibit, whereas phosphatidic acid and sphingomyelin enhance SPCA1a activity. Moreover, SPCA1a is blocked by μM concentrations of commonly used SERCA1a inhibitors thapsigargin (Tg), cyclopiazonic acid (CPA) and 2,5-di-tert-butyl hydroquinone (BHQ). Since tissue-specific targeting of SERCA2b by Tg analogues is considered for prostate cancer therapy, the inhibition of SPCA1a by Tg might represent an off-target risk. We assessed the structure-activity relationship (SAR) of Tg for SPCA1a by in silico modeling, site-directed mutagenesis, and by measuring the potency of a series of Tg analogues. These indicate that Tg and the analogues are bound via the Tg scaffold, but with lower affinity to the same homologous cavity as on the membrane surface of SERCA1a. The lower Tg affinity may depend on a more flexible binding cavity in SPCA1a, with low contributions of the Tg O-3, O-8 and O-10 chains to the binding energy. Conversely, the protein interaction of the Tg O-2 side chain with SPCA1a appears comparable with that of SERCA1a. These differences define a SAR of Tg for SPCA1a distinct from that of SERCA1a, indicating that Tg analogues with a higher specificity for SPCA1a can probably be developed.