Lactate contains a chiral carbon and thus has two optical isomers-d-lactate and 
l-lactate. l-Lactate is the predominant form that is produced by the body and 
can be delivered to the organs. On the other hand, gut microbiota produce both 
isomers, which can then flow into the body. Although both d-lactate and 
l-lactate can contribute to energy metabolism, their potential roles in 
adipocyte differentiation remain to be elucidated. Here, we investigated the 
effects of l-lactate and d-lactate on the differentiation of 3T3-L1 
preadipocytes. Both lactate enantiomers were demonstrated to enhance 
triglyceride accumulation by stimulating the early phase of adipocyte 
differentiation. Notably, d-lactate was more potent than l-lactate in inducing 
triglyceride accumulation. The degree of triglyceride accumulation induced by 
l-lactate was similar to that induced by pyruvate. d-Lactate was more potent 
than l-lactate in increasing the activity of glycerol-3-phosphate dehydrogenase. 
Both lactate enantiomers did not affect cell viability. Moreover, both 
enantiomers upregulated the expression of peroxisome proliferator-activated 
receptor γ, CCAAT/enhancer-binding protein (C/EBP) α, sterol regulatory 
element-binding protein-1c, and fatty acid synthase, with d-lactate exerting 
stronger effects than l-lactate. By contrast, lactate did not influence the 
expression of C/EBPβ and C/EBPδ. d-Lactate significantly increased and l-lactate 
tended to increase p38 MAPK phosphorylation, and the p38 MAPK inhibitor SB203580 
inhibited the stimulation of adipocyte differentiation by d-lactate and 
l-lactate. These findings showed that both lactate enantiomers stimulate 
preadipocyte differentiation, with d-lactate showing more potent effects than 
l-lactate. In addition, our study demonstrated that d-lactate and l-lactate 
exert different effects on physiological events.