17β-Hydroxysteroid dehydrogenase type 3 (17β-HSD3) is expressed at high levels in testes and seminal vesicles and is also present in prostate tissue and involved in gonadal and non-gonadal testosterone biosynthesis. The enzyme is membrane-bound and a crystal structure is not yet available. Selective aryl benzylamine-based inhibitors were designed and synthesized as potential agents for prostate cancer therapeutics through structure-based design, using a previously built homology model with docking studies. Potent, selective, low nanomolar IC50 17β-HSD3 inhibitors were discovered using N-(2-([2-(4-chlorophenoxy)phenylamino]methyl)phenyl)acetamide (1). The most potent compounds have IC50 values of approximately 75nM. Compound 29, N-[2-(1-Acetylpiperidin-4-ylamino)benzyl]-N-[2-(4-chlorophenoxy)phenyl]acetamide has an IC50 of 76nM and 30 N-(2-(1-[2-(4-chlorophenoxy)- phenylamino]ethyl)phenyl)acetamide has IC50 of 74nM. Racemic C-allyl derivative 26 (IC50 of 520nM) was easily formed from 1 in good yield and, to determine binding directionality, its enantiomers were separated by chiral chromatography. Absolute configuration was determined using single crystal X-ray crystallography. Only the S-(+)-enantiomer (32) was active with an IC50 of 370nM. Binding directionality was predictable through our in silico docking studies, giving confidence to our model. Importantly, all novel inhibitors are selective over the type 2 isozyme 17β-HSD2 and show <20% inhibition when tested at 10µM. Lead compounds from this series are worthy of further optimisation and development as inhibitors of testosterone production by 17β-HSD3 and inhibition of prostate cancer cell growth.