Triacylglycerol (TAG) is a microbial oil feedstock for biodiesel production that uses an inexpensive substrate, such as glycerol. Here, we demonstrated the overproduction of TAG from glycerol in engineered Saccharomyces cerevisiae via the glycerol-3-phosphate (G3P) pathway by overexpressing the major TAG synthesis. The G3P accumulation was increased 2.4-fold with the increased glycerol utilization gained by the overexpression of glycerol kinase (GUT1). By overexpressing diacylglycerol acyltransferase (DGA1) and phospholipid diacylglycerol acyltransferase (LRO1), the engineered YPH499 (pGutDgaLro1) strain produced 23.0?mg/L lipids, whereas the YPH499 (pESC-TRP) strain produced 6.2?mg/L total lipids and showed a lipid content that was increased 1.4-fold compared with 3.6% for the wild-type strain after 96?h of cultivation. After 96?h of cultivation using glycerol, the overall content of TAG in the engineered strain, YPH499 (pGutDgaLro1), yielded 8.2% TAG, representing a 2.3-fold improvement, compared with 3.6% for the wild-type strain. The results should allow a reduction of costs and a more sustainable production of biodiesel. Biotechnol. Bioeng. 2013; 110: 343347. (C) 2012 Wiley Periodicals, Inc.