Mutations in polycystins (PC1 or PC2/TRPP2) cause progressive polycystic liver disease (PLD). In PC2‐defective mice, cyclic 3′,5′‐adenosine monophosphate/ protein kinase A (cAMP/PKA)‐dependent activation of extracellular signal‐regulated kinase/ mammalian target of rapamycin (ERK‐mTOR) signaling stimulates cyst growth. We investigated the mechanisms connecting PC2 dysfunction to altered Ca2+ and cAMP production and inappropriate ERK signaling in PC2‐defective cholangiocytes. Cystic cholangiocytes were isolated from PC2 conditional‐KO (knockout) mice (Pkd2flox/−:pCxCreER™; hence, called Pkd2KO) and compared to cholangiocytes from wild‐type mice (WT). Our results showed that, compared to WT cells, in PC2‐defective cholangiocytes (Pkd2KO), cytoplasmic and ER‐Ca2+ (measured with Fura‐2 and Mag‐Fluo4) levels are decreased and store‐operated Ca2+ entry (SOCE) is inhibited, whereas the expression of Ca2+‐sensor stromal interaction molecule 1 (STIM1) and store‐operated Ca2+ channels (e.g., the Orai1 channel) are unchanged. In Pkd2KO cells, ER‐Ca2+ depletion increases cAMP and PKA‐dependent ERK1/2 activation and both are inhibited by STIM1 inhibitors or by silencing of adenylyl cyclase type 6 (AC6). Conclusion: These data suggest that PC2 plays a key role in SOCE activation and inhibits the STIM‐dependent activation of AC6 by ER Ca2+ depletion. In PC2‐defective cells, the interaction of STIM‐1 with Orai channels is uncoupled, whereas coupling to AC6 is maximized. The resulting overproduction of cAMP, in turn, potently activates the PKA/ERK pathway. PLD, because of PC2 deficiency, represents the first example of human disease linked to the inappropriate activation of store‐operated cAMP production. (HEPATOLOGY 2012)