Currently, the complex mechanism of lipid production in microalgal cells is still unclear, and the platform suitable for microalgal genetic transformation is urgent to be established. In this study, the whole genome of a lipid-rich microalgal mutant strain Scenedesmus sp. Z-4 and a lipid-poor wild strain Scenedesmus sp. MC-1 were sequenced, and results revealed that the sequences of 1,256 genes were changed and 148 differential genes related to glucose and lipid metabolism were identified. Especially, gene differentiation of acetyl-CoA carboxylase (ACCase) and phosphoenolpyruvate carboxylase (PEPC) in mutant strain Z-4 and wild strain MC-1, which played key roles in lipid synthesis, were evaluated. Furthermore, to investigate whether mutated ACCase and PEPC genes affect the lipid production, two genes from mutant strain Z-4 were transformed into the expression system of wild strain MC-1. Nine transformants with higher lipid content were successfully obtained, in which the optimal transformant with 28.6% more intracellular lipid than wild strain MC-1 was isolated by overexpression of mutated ACCase gene, demonstrating the important role of ACCase in lipid accumulation of microalgal cells. These results could provide a better understanding of the superior lipid production of mutant strain Scenedesmus sp. Z-4.