Isothiocyanates (ITCs) have gained increasing attention since they have been attributed the merits for the potential beneficial effects of cruciferous vegetable dietary consumption on cancer. The aim of the present study was to determine the cytotoxic effects of 3-butenyl ITC (3-BI) on prostate cancer (PC) cells under in vitro conditions. Two androgen-insensitive human PC cell lines, PC-3 and DU145, were assayed. Cells were cultured in the presence of increasing concentrations of 3-BI (5, 10, 30 and 50 µM) in the absence or presence of the chemotherapeutic drug docetaxel (DOCE) (1 and 2 nM). The cytotoxic effects of these compounds were analyzed using the trypan blue exclusion assay at 24, 48 and 72 h. Apoptosis and migration assays were also performed. The results showed that 3-BI induced a dose-dependent cytotoxic effect on PC-3 cells at 24, 48 and 72 h. These effects were significantly higher than those found with DOCE at 72 h of culture. Moreover, 3-BI also potentiated the effects of DOCE in a dose-dependent manner. Additionally, 3-BI showed inhibition of the migration of PC-3 cells. Nevertheless, 3-BI was not effective in the DU145 PC cell line. These results show a promising role for the 3-BI compound as a co-adjuvant agent in DOCE-based therapy in certain types of PC.