Lactic acid is one of the backbone organic acids in Chinese baijiu. Its content affects both the fermentation process and the flavor and quality of baijiu. However, contribution of different microorganisms to lactic acid synthesis and the metabolism of lactic acid during strong-flavor baijiu fermentation have not yet been clarified. In this study, microbes and pathways involved in lactate synthesis during strong-flavor baijiu fermentation were revealed by RNA-seq. It was found that lactate was synthesized under microaerobic and anaerobic conditions, and both nitrogen and carbon metabolisms were involved. In the process of nitrogen metabolism, Acetobacter malorum, Acetobacter pasteurianus, Komagataeibacter xylinus, Pichia membranifaciens and Saccharomyces cerevisiae synthesized d-lactate. Torulaspora delbrueckii synthesized lactate through homolactic fermentation under microaerobic condition. In the process of strong-flavor baijiu fermentation, lactate is mainly synthesized by Lactobacillus through heterolactic fermentation. Lactobacillus ozensis and Lactobacillus diolivorans were identified as two major lactate producers and they synthesized d-lactate under anaerobic condition. l-lactate could be synthesized by T. delbrueckii, L. ozensis and Lactobacillus acetotolerans. The results demonstrated a comprehensive and clarified mechanism of lactic acid synthesis during strong-flavor baijiu fermentation, and could provide a theoretical basis for regulation of lactic acid synthesis in the process of baijiu fermentation.