Missense mutations in the cystathionine beta-synthase (CBS) gene, such as I278T, are responsible for CBS deficiency, the most common inherited disorder in sulfur metabolism. Expression of human mutant CBS proteins in Saccharomyces cerevisiae reveals that most disease causing mutations severely inhibit enzyme activity and cannot support growth of yeast on cysteine-free media. Here, we show that the osmolyte chemical chaperones glycerol, trimethylamine-N-oxide, dimethylsulfoxide, proline or sorbitol, when added to yeast media, allows growth on cysteine-free media and causes increased enzyme activity from I278T and three other mutant CBS proteins. Rescuable mutants are ones that are predicted to cause a decrease in solvent accessible surface area. The increase in enzyme activity is associated with stabilization of the tetramer form of the enzyme. This effect is not specific to yeast, as addition of the chaperone glycerol resulted in increased I278T activity when the enzyme is produced either in Escherichia coli or in a coupled in vitro transcription/translation reaction. However, no stimulation of specific activity was observed when chaperones were added directly to purified I278T indicating that the presence of chemical chaperones is required during translation. We also found that by mixing different chaperones we could achieve rescue at significantly lower chaperone concentrations. Taken together, our data show that chemical chaperones present during the initial folding process can facilitate proper folding of several mutant CBS proteins and suggest it may be possible to treat some inborn errors of metabolism with agents that enhance proper protein folding.