Yarrowia lipolytica is considered as a promising microbial cell factory for bio-oil production due to its ability to accumulate a large amount of lipid. However, the regulation of lipid metabolism in this oleaginous yeast is elusive. In this study, the MHY1 gene was disrupted, and 43.1% (w/w) intracellular oil based on cell dry weight was obtained from the disruptant M-MHY1, while only 30.2% (w/w) lipid based on cell dry weight was obtained from the reference strain. RNA-seq was then performed to analyze transcriptional changes during lipid biosynthesis after MHY1 gene inactivation. The expression of 1597 genes, accounting for 24.7% of annotated Y. lipolytica genes, changed significantly in the disruptant M-MHY1 during lipid biosynthesis. Differential gene expression analysis indicated that Mhy1p performs multiple functions and participates in a wide variety of biological processes, including lipid, amino acid and nitrogen metabolism. Notably, data analysis revealed increased carbon flux through lipid biosynthesis following MHY1 gene inactivation, accompanied by decreased carbon flux through amino acid biosynthesis. Moreover, Mhy1p regulates the cell cycle, and the cell cycle rate was enhanced in the disruptant M-MHY1. These results suggest that Mhy1p plays critical regulatory roles in diverse aspects of various biological processes, especially in lipid biosynthesis, amino acid and nitrogen metabolism and cell cycle. Our dataset appears to elucidate the crucial role of Mhy1p in lipid biosynthesis and serves as a resource for exploring physiological dimorphic growth in Y. lipolytica.