Lithothamnion sp., a red algae of the Corallinaceae family, when harvested in its calcareous form, is rich in calcium, magnesium and a variety of trace minerals. It is used as a beneficial dietary mineral supplement across the world (Aquamin F). This study was designed to evaluate the acute and sub-chronic toxicity of Lithothamnion sp. according to the Procedure and Methods of Food Safety Toxicological Assessment GB-15193 (China). In an acute toxicity test, mice (n = 20, 10 male, 10 female) were administered a single dose of 10 g/kg BW of Lithothamnion sp. No mortality, or signs of toxicity were observed. In the sub-chronic toxicity arm of the study, SD rats (n = 80, 40 male, 40 female) were randomly divided into four groups with 10 rats in each group and provided pelleted food containing the algae at either 0.00 %, 0.625 %, 1.25 %, 2.50 % inclusion rates for 90 days. Lithothamnion sp. at all inclusion rates did not cause any mortality, and no treatment-related changes were observed in body weight, organ weight, feed consumption, feed utilization rate, urinalysis, hematological and biochemical blood analysis, gross necropsy or histopathologic examinations. In summary, the median lethal dose (LD50) of Lithothamnion sp. was >10 g/kg BW. The no-observed-adverse-effect-level (NOAEL) for female and male rats of Lithothamnion sp. under these experimental conditions was 2.69 g/kg BW and 2.10 g/kg BW respectively.