Betulinic acid and its derivatives are potential bioactive compounds present in nature. This study investigated the biotransformation of betulin to betulinic acid by Cunninghamella blakesleeana cells. LCMS analysis demonstrated that betulin could be transformed into at least five products from cultured C. blakesleeana cells, among which betulinic acid was the most important. The presented method provides an attractive alternative approach to chemical synthesis, because is less time-consuming and more environmentally friendly. C. blakesleeana can transform betulin into potent derivatives with high pharmacological activities.