Inhibition of 17β-HSD2 is an attractive mechanism for the treatment of osteoporosis. We report here the optimization of human 17β-HSD2 inhibitors in the 2,5-thiophene amide class by varying the size of the linker (n equals 0 and 2) between the amide moiety and the phenyl group. While none of the phenethylamides (n = 2) were active, most of the anilides (n = 0) turned out to moderately or strongly inhibit 17β-HSD2. The four most active compounds showed an IC(50) of around 60 nM and a very good selectivity toward 17β-HSD1, 17β-HSD4, 17β-HSD5, 11β-HSD1, 11β-HSD2 and the estrogen receptors α and β. The investigated compounds inhibited monkey 17β-HSD2 moderately, and one of them showed good inhibitory activity on mouse 17β-HSD2. SAR studies allowed a first characterization of the human 17β-HSD2 active site, which is predicted to be considerably larger than that of 17β-HSD1.