Abstract Background and purpose Carbonic anhydrase (CA) 9 expression is induced under hypoxic conditions. Recently we discovered that hypoxia-induced CA9 expression requires an intact unfolded protein response (UPR) pathway. The objective of this study was to evaluate the effect of reduced CA9 expression in UPR-impaired tumor cells on pH regulation and survival under acidic conditions. Materials and methods CA9 expression was assessed in wild-type or UPR-defective U373-MG cells by quantitative-PCR, Western blot, immunofluorescence and flow cytometry. Proliferation and cell survival were measured under acidic conditions in the absence of bicarbonate under ambient CO 2 levels. Results Cell-surface CA9 expression was significantly reduced in UPR-defective U373 cell lines compared to parental U373 cells. This resulted in decreased CA9 activity and extracellular acidification. Differences in CA9 expression and activity were correlated with the ability to tolerate acidic conditions. In addition we observed a strong synergistic effect of anoxia and low pH on cell survival. Conclusions Our data indicate that UPR-impaired U373 cell lines have a defect in pH regulation due to reduced CA9 expression that sensitizes to acidic conditions. We speculate that this defect may contribute to the poor growth of UPR-defective tumors.