Objective: To assess the potential toxicity of hydroalcoholic extract of Allium affine (A. affine) aerial parts after acute and sub-acute administration in female and male Wistar rats. Methods: For acute toxicity assay, animals orally received the limit test dose of 2000 mg/kg of A. affine extract and were observed for 2 weeks. For sub-acute toxicity study, rats were orally treated with 125, 250, and 500 mg/kg/day of the extract over 28 days, and hematological, biochemical, and histological evaluations were then conducted. Results: All rats were alive with normal body weight gain over 14 days, with LD50?2000 mg/kg. No abnormality in body weight changes but significant increases in the relative weight of spleen and lung were detected after administration of the highest dose of extract for 28 days in sub-acute assay. Hematological analysis showed prominent elevations in total white blood cells in male rats and neutrophils count in female rats after exposure to 500 mg/kg of A. affine extract. In biochemical evaluations, significant increases in serum creatinine level (female rats, 250 and 500 mg/kg) and in aspartate aminotransferase (male and female rate, 500 mg/kg) and alanine aminotransferase (male, 250 and 500 mg/kg and female, 500 mg/kg) activities, however, notable decreases in serum blood glucose (male rats, 125 and 500 mg/kg), triglycerides (male rats, 500 mg/kg and female rates, 250 mg/kg), and low-density lipoprotein cholesterol levels (male, 250 mg/kg) were found. Histological examinations presented slight portal inflammation in liver tissue, moderate pneumocyte hyperplasia, congestion and peri-bronchial inflammation in lung tissue, and mild histiocytosis and lymphoid follicular activation in spleen tissue after exposure to 500 mg/kg of A. affine extract in male and female animals. Conclusions: The present investigation reveals the safety of A. affine extract at doses of lower than 250 mg/kg in rats and monitoring of lung, spleen, and liver functions is suggested during excessive and prolonged uses.