The yeast Candida glycerinogenes produces a high yield of glycerol only in response to a medium-osmotic stress, but little is known about the relationship between osmoadaptation and glycerol metabolism. The CgTPI gene encoding triose phosphate isomerase of C. glycerinogenes was cloned and sequenced, and its functionality was confirmed by complementation of Saccharomyces cerevisiae tpi1 Delta. The roles of CgTpip in the glycerol biosynthesis and the osmoadaptation were investigated. Unlike S. cerevisiae tpi1 Delta and Klyuveromyces lactis tpi1 Delta, the mutant lacking CgTPI significantly decreased the rate of glucose consumption and the glycerol yield. Furthermore, the mutants decreased osmotolerance to glucose and NaCl. The results suggest that CgTPI might be crucial for a high yield of glycerol by C. glycerinogenes. The inhibition of glycerol biosynthesis might be related to the reduced ability of osmoadaptation to high external osmolarity. To our knowledge, this is the first report that inactivation of a yeast TPI gene inhibits the biosynthesis of glycerol.