The differentiation of 3T3-L1 cells into adipocytes involves the activation of an organized system of obesity-related genes, of which those encoding CCAAT/enhancer-binding proteins (C/EBPs) and the Wnt-10b protein may play integral roles. In a previous study of ours, we found that a specific peptide found in tuna (sequence D-I-V-D-K-I-E-I; termed TP-D) inhibited 3T3-L1 cell differentiation. In the present study, we observed that the expression of expression of C/EBPs and Wnt-10b was associated with obesity. The initial step of 3T3-L1 cell differentiation involved the upregulation of C/EBP-α expression, which in turn activated various subfactors. An upstream effector of glycogen synthase kinase-3β (GSK-3β) inhibited Wnt-10b expression in 3T3-L1 adipocytes. In a previous study of ours, we sequenced the tuna peptide via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and quadrupole time-of-flight mass spectrometry (Q-TOF MS/MS) and confirmed the anti-obesity effects thereof in 3T3-L1 adipocytes. In the present study, we demonstrate that TP-D inhibits C/EBP and promotes Wnt-10b mRNA expression, thus activating the Wnt pathway. The inhibition of lipid accumulation was measured using a glucose and triglyceride (TG) assay. Our results confirmed that TP-D altered the expression levels of C/EBP-related genes in a dose-dependent manner and activated the Wnt signaling pathway. In addition, we confirmed that total adiponectin and high-molecular weight (HMW) adiponectin levels were reduced by treatment with TP-D. These data indicate that TP-D inhibits adipocyte differentiation through the inhibition of C/EBP genes and the subsequent activation of the Wnt/β-catenin signaling pathway.