In recent years, bio-based production of free fatty acids from renewable resources has attracted attention for their potential as precursors for the production of biofuels and biochemicals. In this study, the oleaginous yeast Yarrowia lipolytica was engineered to produce free fatty acids by eliminating glycerol metabolism. Free fatty acid production was monitored under lipogenic conditions with glycerol as a limiting factor. Firstly, the strain W29 (Delta gpd1), which is deficient in glycerol synthesis, was obtained. However, W29 (Delta gpd1) showed decreased biomass accumulation and glucose consumption in lipogenic medium containing a limiting supply of glycerol. Analysis of substrate utilization from a mixture of glucose and glycerol by the parental strain W29 revealed that glycerol was metabolized first and glucose utilization was suppressed. Thus, the Delta gpd1 Delta gut2 double mutant, which is deficient also in glycerol catabolism, was constructed. In this genetic background, growth was repressed by glycerol. Oleate toxicity was observed in the Delta gpd1 Delta gut2 Delta pex10 triple mutant strain which is deficient additionally in peroxisome biogenesis. Consequently, two consecutive rounds of selection of spontaneous mutants were performed. A mutant released from growth repression by glycerol was able to produce 136.8 mgL(-1) of free fatty acids in a test tube, whereas the wild type accumulated only 30.2 mgL(-1). Next, an isolated oleate-resistant strain produced 382.8 mgL(-1) of free fatty acids. Finely, acyl-CoA carboxylase gene (ACC1) over-expression resulted to production of 1436.7 mgL(-1) of free fatty acids. The addition of dodecane promoted free fatty acid secretion and enhanced the level of free fatty acids up to 2033.8 mgL(-1) during test tube cultivation.