Oyster mushrooms are popular and physiologically beneficial to human health. This study was conducted to investigate the effect of dietary supplementation of oyster mushrooms such as Pleurotus ostreatus, P. pulmonarius, and P. citrinopileatus on high cholesterol fed rats. Five-week old female Sprague-Dawley albino rats were divided into three groups of 8 rats each. Feeding a diet containing a 5% fruiting body powder of Pleurotus species to high cholesterol fed rats reduced plasma total cholesterol (TG), triglyceride (TG), low-density lipoprotein (LDL). Feeding each oyster mushroom also significantly reduced body weight in high cholesterol fed rats. However, it had no adverse effects on plasma albumin, creatinine, blood urea nitrogen, uric acid, glucose, total protein or enzyme profiles such as glutamate oxalate transaminase (GOT), glutamate pyruvate transaminase (GPT), and alkaline phosphatase (ALP) which are related to kidney function. Feeding each mushroom increased total lipid and cholesterol excretion in feces. A histological study of hepatic cells by conventional red O staining showed normal findings in mushroom-fed hyperlipidemic rats. These results suggested that diet supplementation of 5% fruiting body power of Pleurotus species provided health benefits by acting on the atherogenic lipid profile in high cholesterol fed rats.