A new androsterone derivative bearing a 16β-picolyl moiety (compound 5; FCO-586-119) was synthetized in four steps from the lead compound 1 (RM-532-105). We measured its inhibitory activity on 17β-HSD3 using microsomal fraction of rat testes as well as transfected LNCaP[17β-HSD3] cells. We then assessed its metabolic stability as well as its cytotoxic effect against a panel of cancer cell lines. The addition of a picolyl moiety at C-16 of RM-532-105 steroid core improves the 17β-HSD3 inhibitory activity in the microsomal fraction of rat testes, but not in whole LNCaP[17β-HSD3] cells. Interestingly, this structural modification enhances 3-fold the metabolic stability in conjunction with a significant cytotoxic effect against pancreatic, ovarian, breast, lung, and prostate cancer cells. Because the inhibitory activity data against 17®-HSD3 suggested that both steroid derivatives are non-competitive inhibitors, we performed docking and molecular dynamics simulations using a homology model of this membrane-associated enzyme. The results of these simulations revealed that both RM-532-105 (1) and FCO-586-119 (5) can compete for the cofactor-binding site displaying better binding energy than NADP.