Acceleration of cell growth is obviously useful in cell culture for cell biology and clinical treatment. The present study investigated the effects of carbonic anhydrase (CA) on cell growth, since CA can change pH with balance between CO₂ and HCO₃^-. Human gastric cancer cells (MK-1 cells) were cultured with or without acetazolamide (ACTZ, a CA inhibitor), for 2 or 7 days. Cell proliferation was observed after 2 days culture using 3-(4,5-dimethyl-2- thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay which base on the reduction of the soluble yellow MTT tetrazolium salt to a blue insoluble formazan product by mitochondrial succinic dehydrogenase, only the alive cells can reduce the soluble yellow MTT tetrazolium salt to a blue insoluble formazan product, while colony formation was examined after 7 days culture. In another experiment, cells were loaded with 2’,7’-bis(2-carboxyethyl)-5(6)–carboxyfluorescein (BCECF)-AM, a fluorescent pH indicator, and fura-2-AM, a calcium indicator. Intracellular pH (pHi) and Ca²⁺ (Ca²⁺i) were then measured using microspectrofluorometry. Statistical analysis was performed using ANOVA for comparison of MTT assay and colony formation vs. ACTZ. For comparison of pHi and Ca²⁺i, student`s unpaired t-tests was used. Compared to controls, a 5% increase in cell proliferation and 40% increase in colony formation were observed for cells cultured with ACTZ (p<0.01). The ACTZ group displayed lower pHi than the control group, with a concomitant increase in Ca²⁺i (p< 0.01). Decreases in CA activity resulting from the use of ACTZ could promote cell growth, due to decreased pHi. However, ACTZ could not change Ca²⁺i.