Indole-3-carbinol (I3C) is an active component of cruciferous vegetables and markedly inhibits the growth of a variety of tumors. However, its role in laryngeal cancer remains obscure. The aim of the present study was to elucidate the possible mechanisms whereby I3C influences Hep-2 laryngeal cancer cell proliferation and apoptosis. Treatment with I3C dose-dependently and significantly inhibited Hep-2 cell proliferation, and at doses of 100 and 150 µM, I3C induced cell morphological changes and promoted apoptosis. Following treatment of Hep-2 cells with I3C, we found that the protein expression of phosphatidylinositol-3-kinase (PI3K) p110α, PI3K p110β, PI3K class III, p-PDK1, Akt, p-Akt and the downstream signaling proteins p-c-Raf and GSK3-β were significantly downregulated. Additionally, tumor-bearing mouse models were constructed using BALB/c nude mice. The mice were subdivided into groups: pretreated with I3C, or treated with I3C or an untreated control group. After 8 weeks, mice pretreated or treated with IC3 developed smaller tumors compared to the untreated control group, and the protein expression of PI3K p110α, PI3K class III, Akt, p-Akt and the downstream signaling proteins p-c-Raf and GSK3-β in the tumors were significantly downregulated. Furthermore, no harmful side effect were observed in the heart, liver and kidney of the I3C-treated nude mice. In conclusion, I3C inhibited proliferation and induced the apoptosis of laryngeal tumor cells both in vivo and in vitro, and exhibited low toxicity to normal cells. The inhibitory effects noted with I3C treatment may depend on decreased phosphatidylinositol-3 kinase/serine-threonine kinase (PI3K/Akt) expression. This approach may be applied to the clinical treatment of laryngeal tumors and in drug screening.