Trichosanthin (TCS) is a type I ribosome--inactivating protein, which inhibits cell viability in human epithelial type 2 (HEp-2) and AMC-HN-8 human laryngeal epidermoid carcinoma cells. Although TCS is a potential chemotherapeutic agent, its mechanism of action remains to be elucidated. In the present study, HEp-2 and AMC-HN-8 cells were treated with different concentrations of TCS combined with or without cisplatin. After 5 days of successive treatment, different experimental groups were detected using a cell counting kit-8 and the collected supernatants were analyzed using a lactate dehydrogenase kit. Flow cytometric assays were performed to detect apoptosis and cell cycle arrest in the HEp-2 and AMC-HN-8 cells, reverse transcription quantitative polymerase chain reaction was performed to detect the levels of p27, p21WAF and western blot analysis was performed to detect changes in c-Jun N-terminal protein kinase (JNK)/phosphorylated (phospho)-JNK, p38/phospho-p38, extracellular signal-regulated kinase (ERK)/phospho-ERK, caspase-3 and caspase-9 in the HEp-2 and AMC-HN-8 cancer cells. TCS significantly inhibited the cell viability of the HEp-2 and AMC-HN-8 cells, independently of necrosis. TCS induced apoptosis and increased the percentage of HEp-2 and AMC-HN-8 cells in the S-phase of the cell cycle. In addition, the JNK/mitogen-activated protein kinase (MAPK) pathway was activated by TCS in the HEp-2 and AMC-HN-8 cells. Low concentrations of TCS also induced apoptosis and S-phase cell cycle arrest in the HEp-2 and AMC-HN-8 cells. The antitumor effects of TCS may be associated with JNK/MAPK activation.