To extract, isolate and identify active constituents in Bufo melanostictus Schneider, and to study their anti-proliferative and apoptosis-inducing effects on human gastric cancer MGC803 cells. Chemical constituents were isolated and purified by repeated silica gel column chromatography, ODS reverse phase column chromatography, preparative liquid chromatography and recrystallization, and structurally elucidated based on physicochemical properties & spectral data of compounds and reference literatures. Anti-proliferative effects of different concentrations of Bufo melanostictus Schneider extracts on gastric cancer MGC803 cells were detected by MTT, and their morphological changing effects on these cells were observed by microscopy. Four compounds were isolated from the skin of Bufo melanostictus Schneider. The anti-proliferative effects of Bufo melanostictus Schneider extracts on gastric cancer MGC803 cells were enhanced with increasing concentration and action time. 72 h after action of test solution in the high-dose group, proliferation inhibition rate of MGC-803 cells reached 77.5%. Under the microscope, firmly adherent MGC-803 cells with intact membranes were observed in the control group; cells were bright and clear, had good refraction, and were fully spread; uniform cytoplasmic distribution was observed as well. In various treatment groups, cell density became gradually sparse with increasing drug concentration, and cell surfaces were wrinkled. In the high-dose group, most cells were disrupted; cell morphology was not intact; cell refraction decreased; most cells were detached; chromatins were condensed, compacted and split into blocks; and the number of adherent cells decreased. Active constituents in Bufo melanostictus Schneider can induce apoptosis of gastric cancer MGC803 cells.