CYP105A1 from Streptomyces griseolus converts vitamin D3 to its biologically active form, 1α,25-dihydroxy D3. R73A/R84A mutation enhanced the 1α- and 25-hydroxylation activity for D3, while M239A mutation generated the 1α-hydroxylation activity for vitamin D2. In this study, the stability of six CYP105A1 enzymes, including five variants (R73A/R84A, M239A, R73A/R84A/M239A ( = TriA), TriA/E90A, and TriA/E90D), were examined. Circular dichroism analysis revealed that M239A markedly reduces the enzyme stability. Protein fluorescence analysis disclosed that these mutations, especially M239A, induce large changes in the local conformation around Trp residues. Strong stabilizing effect of glycerol was observed. Non-denaturing PAGE analysis showed that CYP105A1 enzymes are prone to self-association. Fluorescence analysis using a hydrophobic probe 8-anilino-1-naphthalenesulfonic acid suggested that M239A mutation enhances self-association and that E90A and E90D mutations, in cooperation with M239A, accelerate self-association with little effect on the stability.