Lentinus edodes is an edible mushroom studied for use, or as an adjunct, in the prevention of illnesses such as hypertension, hypercholesterolemia, diabetes, and cancer. Despite the functional properties of L. edodes, the doses commonly reported in experimental studies are much higher than those actually consumed. Thus, we aimed to establish the optimum intake levels of L. edodes in vivo. Four groups of male Wistar rats received dry and powdered L. edodes reconstituted in water for 30 days: control (water only), L. edodes 100 mg/kg, L. edodes 400 mg/kg, and L. edodes 800 mg/kg. Biochemical and hematological parameters were assessed using commercial kits. Antioxidant parameters were quantified spectrophotometrically. Neither cholesterol, triglycerides, glucose, nor transaminase activity was different among any of the L. edodes concentrations. However, fructosamine concentrations were significantly decreased in groups consuming L. edodes at 100 or 400 mg/kg. A significant decrease in hemoglobin concentration was found in the 400 and 800 mg/kg/day L. edodes groups, and leukopenia occurred in rats that consumed L. edodes 800 mg/kg/day compared with the control group. L. edodes at 100 and 400 mg/kg increased amounts of reduced glutathione compared with the control group. L. edodes was effective as an antioxidant at 100 and 400 mg/kg, but at 400 and 800 mg/kg some disturbances were observed, such as reductions in hemoglobin and leukocytes. In summary, this study has potential benefits for scientific development because the safe daily intake of L. edodes (at 100 mg/kg) is, to our knowledge, reported for the first time in a preclinical study.