Objective To investigate the transcriptional regulation of cellobiose synthase (CBS) in Rhizopus stolonifer. Results Transcription factor XYR1 was identified as responsible for the activation of cbs. In comparison with wild-type R. stolonifer, the deletion of XYR1 resulted in transcriptional down-regulation of cbs by approximately 40%, while XYR1 over-expression increased cbs transcription up to 175%. The highest FPA activity (1.8 IU/ml) was obtained in the XYR1-overexpressing strain OExyr1 cultivated in a 2% (m/V) glucose media, corresponding to a 96% increase compared with that of the parent strain (0.92 IU/ml). Moreover, cellulase synthesis was inhibited after cbs-inactivation mutation in OExyr1. Conclusion XYR1 directly activates the transcription of cbs to promote cellulase production in R. stolonifer utilizing glucose as a substrate.