S100A4 is a small Ca2+ binding protein that belongs to the S100 family and is involved in a number of cellular functions, including cell cycle control, proliferation, apoptosis, and has a significant role in angiogenesis and neurite extension. However, the exact function and mechanism of S100A4 in laryngeal carcinogenesis remains to be elucidated. The present study was designed to investigate the potential use of RNA interference to inhibit S100A4 expression and activation, as well as the subsequent effect on human laryngeal cancer cell growth and apoptosis. The present study demonstrated that knockdown of S100A4 decreased the proliferation and growth of the human laryngeal cancer Hep‑2 cell line. The percentages of the apoptotic cells were 4.23±1.22, 4.92±1.85 and 11.70±4.02% in the control, negative control and S100A4 short hairpin RNA (shRNA) groups, respectively, indicating significant differences among the different groups. The S100A4‑mediated induction of apoptosis was demonstrated to be associated with the activation of caspase‑3, caspase‑8 and caspase‑9. Intratumoral injection of S100A4‑shRNA inhibited tumor growth in nude mice. Thus, knockdown of S100A4 inhibited the progression of laryngeal squamous carcinoma, decreased proliferation and promoted apoptosis. S100A4 is a potential candidate for therapeutic targeting of laryngeal carcinoma cells.