Since glycerol is one of the precursors for lipids synthesis in oleaginous yeasts [21], the use of it as a carbon source would promote the synthesis rate of lipids. In this study, the crude glycerol from biodiesel plant was used as the sole carbon source for the production of value-added lipids and carotenoids by R. glutinis TISTR 5159. A suitable surfactant was first screened for enhanced production of lipids and carotenoids. The effects and optimum levels of glycerol concentration, C/N ratio and surfactant concentration were determined for maximizing biomass, lipid content and production of carotenoids using RSM. The culture conditions, including pH control and aeration rate, were optimized in a stirred tank bioreactor. The present study also developed a fed-batch process to enhance the production of lipids and carotenoids. Furthermore, the fuel properties of yeast lipids obtained were also determined.