Pleurotus eryngii var. ferulae (PEF) is traditionally used in the prevention and treatment of lifestyle-related diseases. In this study, we investigated the ability of PEF extract to prevent obesity and metabolic diseases and explored the underlying mechanism. Mice were fed a high-fat diet (HFD) containing PEF extract for 12 weeks, and their body weight, adipose tissue and liver weights, and lipid profiles and blood glucose levels, were monitored. Fecal triglyceride (TG) levels were also measured and olive oil-loading tests were performed. Furthermore, the effect of PEF extract on pancreatic lipase (PL) activity was examined in vitro. Treatment with PEF extract for 12 weeks resulted in a significant decrease in the HFD-induced increases in body weight, white adipose tissue weight, liver weights, and lipid profiles, and improved glucose tolerance and insulin sensitivity. To assess the mechanism underlying the effect of PEF extract on obesity and diabetes, we investigated its role in inhibiting lipid absorption. Consumption of an HFD containing PEF extract significantly increased the TG level in feces compared with the controls, suggesting inhibition of TG absorption in the digestive tract. Furthermore, PEF extract suppressed the increase in serum TG levels resulting from oral administration of a lipid emulsion to mice, confirming inhibition of TG absorption. Moreover, PEF extract inhibited PL activity in vitro. Our combined results indicate that the anti-obesity and antidiabetic effect of PEF extract in mice fed an HFD may be caused by inhibition of lipid absorption as a result of reduced PL activity.