Mai Tong Fang (MTF), a Chinese herbal combination, has been used for the treatment of diabetic nephropathy in traditional medical clinics in China. However, the anti-adipogenic and anti-hyperglycemic effects of MTF have not been fully elucidated, so this study explored these pharmacological activities in 3T3-L1 adipocytes and ob/ob mice, respectively, of the water fraction of milkvetch root, salviae miltiorrhizae and mulberry as key components of MTF. MTF was found to inhibit adipogenesis and triglyceride accumulation in 3T3-L1 adipocytes. Oral administration of MTF in ob/ob mice for 8 weeks, exhibited positive controls on blood glucose and body weight, and further improved glucose tolerance according to an oral glucose tolerance test. Importantly, MTF extract alleviated fat deposition and ballooning degeneration in liver tissue and blocked the increase of adipocyte size in adipose tissue from treated ob/ob mice. These results indicated that the extract of key components in the traditional Chinese prescription MTF continue a potent anti-adipogenic and glucose-lowering agent.