In previous study we isolated a gram-positive bacterial strain, designated Niu-O16, from bovine rumen gastric juice. The growing cells of bacterial strain Niu-O16 is capable of biotransforming isoflavone daidzein into dihydrodaidzein efficiently under anaerobic conditions. In this study we investigated the optimal bioconversion conditions for the resting cells of bacterial strain Niu-O16 to convert daidzein into dihydrodaidzein. Single factor test showed that the optimal conditions for the initial pH of phosphate buffer, the concentration of the resting cell and the concentration of the substrate daidzein were 6.0–8.0, 32–64 mg/mL (wet weight) and 0.8~1.2 mmol/L, respectively. Orthogonal experiments were used to determine the optimal combination of the resting cell concentration, substrate concentration and biotransformation time. The results showed that the optimal combination included resting cell concentration 32 mg/mL, substrate concentration 0.8 mmol/L and the biotransformation time 24 h. Furthermore, the biotransformation kinetics under optimal conditions were studied, under which conditions the highest bioconversion rate was 63.9% in the resting cell system. The results might provide information for resting cell biotransforming of anaerobes as well as its industrial application.