Microalgae have long been considered as potential biological feedstock for the production of wide array of bioproducts, such as biofuel feedstock because of their lipid accumulating capability. However, lipid productivity of microalgae is still far below commercial viability. Here we identified a glucose-6-phosphate dehydrogenase from the oleaginous microalga Nannochloropsis oceanica and heterologously expressed it in the green microalga Chlorella pyrenoidosa to characterize its function in the pentose phosphate pathway. We found that the G6PD enzyme activity towards NADPH production was increased by 2.19-fold in engineered microalgal strains. Lipidomic analysis revealed an up to 3.09-fold increase of neutral lipid content in the engineered strains, and lipid yield was gradually increased throughout the cultivation phase and saturated at the stationary phase. Moreover, cellular physiological characteristics including photosynthesis and growth rate were not impaired. Collectively, these results revealed the pivotal role of glucose-6-phosphate dehydrogenase from N. oceanica in NADPH supply, demonstrating that provision of reducing power is crucial for microalgal lipogenesis and could be a potential target for metabolic engineering. This article is protected by copyright. All rights reserved.