Lipid production by Saccharomyces cerevisiae was improved by overexpression of the yeast diacylglycerol acyltransferase Dga1p lacking the N-terminal 29 amino acids (Dga1a dagger Np), which was previously found to be an active form in the a dagger snf2 mutant. Overexpression of Dga1a dagger Np in the a dagger snf2 mutant, however, did not increase lipid content as expected, which prompted us to search for a more suitable strain in which to study the role of Dga1a dagger Np in lipid accumulation. We found that the overexpression of Dga1a dagger Np in the a dagger dga1 mutant effectively increased the lipid content up to about 45 % in the medium containing 10 % glucose. The high lipid content of the transformant was dependent on glucose concentration, nitrogen limitation, and active leucine biosynthesis. To better understand the effect of dga1 disruption on the ability of Dga1a dagger Np to stimulate lipid accumulation, the a dagger dga1-1 mutant, in which the 3'-terminal 36 bp of the dga1 open reading frame (ORF) remained, and the a dagger dga1-2 mutant, in which the 3'-terminal 36 bp were also deleted, were prepared with URA3 disruption cassettes. Surprisingly, the overexpression of Dga1a dagger Np in the a dagger dga1-1 mutant had a lower lipid content than the original a dagger dga1 mutant, whereas overexpression in the a dagger dga1-2 mutant led to a high lipid content of about 45 %. These results indicated that deletion of the 3' terminal region of the dga1 ORF, rather than abrogation of genomic Dga1p expression, was crucial for the effect of Dga1a dagger Np on lipid accumulation. To investigate whether dga1 disruption affected gene expression adjacent to DGA1, we found that the overexpression of Esa1p together with Dga1a dagger Np in the a dagger dga1 mutant reverted the lipid content to the level of the wild-type strain overexpressing Dga1a dagger Np. In addition, RT-qPCR analysis revealed that ESA1 mRNA expression in the a dagger dga1 mutant was decreased compared to the wild-type strain at the early stages of culture, suggesting that lowered Esa1p expression is involved in the effect of dga1 disruption on Dga1a dagger Np-dependent lipid accumulation. These results provide a new strategy to engineer S. cerevisiae for optimal lipid production.