Vitamin D-dependent rickets type 1 (VDDR-I) is caused by mutation in CYP27B1. The glycine residue at codon 102 is not conserved between human (G(102)) and rodent (S(102)). G102E mutation results in 80% reduction in its enzymatic activity but PolyPhen predicts benign change. It is not known whether G102S has any damaging effect on 1α-hydroxylase activity. We investigated the effect of CYP27B1 (G102S) on its enzymatic activity and compared mutation prediction accuracy for all known CYP27B1 mutations among three free online protein prediction programs: PolyPhen, PolyPhen-2, and PSIPRED. G102S has no damaging effect on 1α-hydroxylase activity. G102D retained 30% enzymatic activity. All three programs correctly predicted damaging change for G102D. PolyPhen predicted benign change for G102S, whereas PolyPhen-2 and PSIPRED indicated possible damaging effect. Among 24 reported damaging mutations, PSIPRED, PolyPhen-2, and PolyPhen achieved 100%, 91.7% (22/24), and 75% (18/24) accuracy rate, respectively. The residues of incorrectly predicted mutations were not conserved. We conclude that G102D resulted in a significant reduction in 1α-hydroxylase activity, whereas G102S did not. PSIPRED and PolyPhen-2 are superior to PolyPhen in predicting damaging mutations.