Sutherlandia frutescens methanol extract (SFE) showed an antiproliferative effect on the human prostate cancer cell lines PC-3 and LNCaP, and mouse prostate cell line TRAMP-C2 (IC50 = 167 μg/mL for PC-3, 200 μg/mL for LNCaP, and 100 μg/mL for TRAMP-C2) associated with a dose-dependent inhibition of the Gli-reporter activity in Shh Light II and TRAMP-C2QGli cells treated with SFE. This evidence concerns the gene GLI1 and prostate carcinoma.