Shiitake mushrooms have been highly regarded as possessing enormous nutritive and medicinal values. No clinical studies have yet investigated the effect of shitake supplementation on the health of horses. The aim of this study was to evaluate the effect of shiitake mushroom supplementation on the morphological and biochemical blood properties in horses. A total of 17 adult horses were divided into two groups: supplemented and control. The supplemented group was fed 60 g of shiitake mushrooms per day for 5 months. Blood samples were collected in five sessions. Blood morphological analysis showed higher levels of lymphocytes in session 3 and monocytes in session 4 in the supplemented group. In addition, basophils, hemoglobin, and hematocrit were elevated compared to the control group. Biochemical analysis showed that the shiitake mushrooms affected a large number of parameters. In particular, alkaline phosphatase was found to be the most sensitive to shitake mushroom supplementation, for which the statistical differences were significant for sessions 2, 4, and 5. Furthermore, calcium was found to be affected by supplementation only in session 4, and gamma-glutamyl transferase in session 2. In addition, the bilirubin and glucose levels were lower in the supplemented group, and the albumin/globulin ratio was higher compared to the control group. The differences between the supplement and the control group in various sessions suggest that shiitake mushrooms are a beneficial nutritional supplement for horses.