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.