An increase in hepatic triglyceride (TG) contents usually results in non-alcoholic fatty liver disease (NAFLD) and related metabolic diseases. However, the mechanisms underlying perturbations of hepatic TG homeostasis remain largely unknown. Here, we showed that MicroRNA-124 was up-regulated in the livers of C57BL/6 mice fed a short-term high-fat-diet (HFD). Adenoviral overexpression of miR-124 in C57BL/6 mice led to accumulation of excessive triglycerides and up-regulation of lipogenic genes in the liver. We further identified tribbles homolog 3 (TRB3) as a direct target of miR-124. AKT signaling, which is negatively regulated by TRB3, was enhanced by miR-124 overexpression. Moreover, restoration of TRB3 expression markedly abolished the effect of miR-124 on hepatic TG metabolism. Therefore, our findings revealed that miR-124 played a role in mediating high-fat-diet induced TG accumulation in the liver.