Asperlin is a marine-derived, natural product with antifungal, anti-inflammatory and anti-atherosclerotic activities. In the present study, we showed that asperlin effectively prevented the development of obesity in high-fat diet (HFD)-fed mice. Oral administration of asperlin for 12 weeks significantly suppressed HFD-induced body weight gain and fat deposition without inhibiting food intake. Hyperlipidemia and liver steatosis were also substantially ameliorated. A respiratory metabolism monitor showed that asperlin efficiently increased energy expenditure and enhanced thermogenic gene expression in adipose tissue. Accordingly, asperlin-treated mice showed higher body temperature and were more tolerant of cold stress. Meanwhile, asperlin also increased the diversity and shifted the structure of gut microbiota. Oral administration of asperlin markedly increased the relative abundance of Bacteroidetes, leading to a higher Bacteroidetes-to-Fimicutes ratio. The HFD-induced abnormalities at both phylum and genus levels were all remarkably recovered by asperlin. These results demonstrated that asperlin is effective in preventing HFD-induced obesity and modulating gut microbiota. Its anti-obesity properties may be attributed to its effect on promoting energy expenditure.