Abstract: Actinomyces CXX-10 is capable of 25-hydroxylating of vitamin D 3 . The effect of surfactant such as Tween-80, Triton-100, glycerin, sodium dodecyl sulfate (SDS) , hexadecyl trimethyl ammonium Bromide (CTAB) cooperate with β-cyclodextrin on productivity of 25 - (OH) D 3 was analysised in this paper. And then optimization of condition of most suitable combination of surfactant and β-cyclodextrin were promoted. The optimal conditions were as follows:0.04% Tween-80 and 0.5% β-cyclodextrin and V D 3 were added after 3d of culture, and other culture condition were 28℃, 180r/min, 7d.