Zygosaccharomyces rouxii was mostly used in high-salt liquid fermentation of soy sauce. To better understand the osmo-adaption mechanism, two key salt-tolerance genes GPD1 coding for glycerol-3-phosphate dehydrogenase and FPS1 coding for a putative glycerol transporter were evaluated in the wild-type Z. rouxii (S) and a higher salt-tolerant mutant strain Z. rouxii 3-2 (S3-2) previously constructed. It was found that several mutations occurred in ZrGPD1 and ZrFPS1 in S3-2 compared with the control strain S. The mutation of ZrGPD1 in S3-2 resulted in the increase of transcription level of ZrGPD1 compared with the control. At the same time, the mutation of ZrFPS1 resulted in the decrease of transcription level of ZrFPS1. In addition, overexpression of S3-2GPD1 and S3-2FPS1 in Saccharomyces cerevisiae could cause the stronger salt tolerance compared to SGPD1 and SFPS1, respectively. The results suggested the improvement of salt tolerance in S3-2 was due to the increase of glycerol contents, which was resulted from the increase of transcription level of ZrGPD1 and the decrease of transcription level of ZrFPS1.