This study was to investigate the effect of phenethyl isothiocyanate (PEITC), a constituent of many edible cruciferous vegetables, on the expression of α- and β-tubulins, which are the main components of microtubules in prostate cancer cells. Flow cytometry, light microscopy and western blot were used to study the cell cycle distribution, morphology changes and the expression of α- and β-tubulins in prostate cancer cells treated with PEITC. The results showed that PEITC-induced G2–M cell phase arrest and inhibited the expression of α- and β-tubulin proteins in a number of human prostatic carcinoma cell lines. Further, it is showed that this inhibitory effect could be reversed by antioxidant N-acetyl cysteine and proteasome inhibitor MG132. Finally, it is concluded that PEITC inhibited the expression of α- and β-tubulins in prostate cancer cells, which is at least related to the oxygen reaction species and protein degradation.