Dissolved oxygen (DO) supply plays essential roles in microbial organic acid production. Candida glabrata, as a dominant strain for producing pyruvic acid, principally converts glucose to pyruvic acid through glycolysis. However, this process relies excessively on high extracellular DO content. In this study, in combination with specific motif analysis of gene promoters, hypoxia-inducible factor 1 (HIF1) was engineered to improve the transcription level of some enzymes related to pyruvic acid synthesis under low DO level and directly led to increased pyruvic acid production and glycolysis efficiency. Moreover, the intracellular stability of HIF1 was further optimized from different aspects to maximize pyruvic acid accumulation. Finally, the pyruvic acid titer in a 5-L batch bioreactor with 10% DO level reached 53.1 g/L. As pyruvic acid is involved in the biosynthesis of various products, these findings suggest that HIF1-enabled regulation method has significant potential for increasing the synthesis of other chemicals in microorganisms.