Streptomycin is a common antibiotic used in culture media. It is also a known blocker of stretch-activated and mechanosensitive ion channels in neurons and cardiac myocytes. But very little information is available on its effect in the regulation of epithelial ion channels. Osmotic swelling is a kind of mechanical stretch. The opening of stretch-activated Ca 2+ channels contributes to hypotonicity-induced Ca 2+ influx which is necessary for the activation of volume-regulated Cl − channels in human cervical cancer cells. This study aimed to investigate the role of streptomycin in cell volume regulation. Treatment of cervical cancer SiHa cells with streptomycin and its analogues (gentamicin and netilmicin) did not affect the basal cytosolic Ca 2+ ([Ca 2+] i) level. But it attenuated the hypotonicity-stimulated increase of [Ca 2+] i in a dose-dependent manner with half-maximal inhibitory concentrations (IC 50) of 25, 90 and 200 μM for streptomycin, gentamicin and netilmicin, respectively, when measured at room temperature. In contrast, under free extracellular Ca 2+ condition, hypotonic stress only induced a small, progressive increase of [Ca 2+] i, while 500 μM streptomycin did not affect this Ca 2+ signaling. Streptomycin and its analogues (gentamicin and netilmicin) also inhibited the activation of volume-regulated Cl − channels in a dose-dependent manner with IC 50 of 30, 95 and 250 μM at room temperature, respectively. Chronic culture with 50 μM streptomycin downregulates the activity of volume-regulated Cl − channels and retards the process of regulatory volume decrease in SiHa cells and MDCK cells. We suggest that using cells chronically cultured with streptomycin to study epithelial ion channels risks studying cellular and molecular pathology rather than physiology.