The sole effect of either saturated fatty acid or moderate ethanol consumption on SLC2A4 (GLUT4) expression is widely reported but the combined effects of them remain obscure. Here, we observed their combined effects on SLC2A4 expression, explored the underlying mechanism mediated by AMP-activated protein kinase alpha (PRKAA2) and myocyte enhancer factor 2 (MEF2) both in vivo and in vitro. In the in vivo experiments, 36 male Wistar rats, divided into three groups, were fed with normal diet, high-fat (HF) diet, or HF diet plus ethanol for 22 weeks. We measured the expressions of total-PRKAA2 (T-PRKAA2), phosphorylated-PRKAA2 (pPRKAA2, activated form of PRKAA2), MEF2, and SLC2A4 in epididymal adipose tissues. In the in vitro experiments, primary adipocytes, isolated from normal Wistar rats, were incubated in the presence or absence of palmitate, ethanol, and compound C (an PRKAA2 inhibitor) for 1 h. Thereafter, T-PRKAA2, pPRKAA2, MEF2, and SLC2A4 expressions were measured. We found that both HF diet and in vitro exposition to palmitate impaired SLC2A4 expression in rat adipocytes with a parallel reduction in PRKAA2 activation and MEF2 expression. This impairment was reversed by ethanol administration. We further demonstrated that ethanol-mediated PRKAA2 activation stimulates MEF2 and SLC2A4 expressions in adipocytes, as evidenced by compound C blockade of these effects. In summary, long-term moderate ethanol consumption reversed the adverse effect of saturated fatty acid on SLC2A4 expression in adipocytes, which was likely to be a result of PRKAA2 activation and subsequent up-regulation of MEF2 and SLC2A4 expressions.