Context: The genus Primula (Primulaceae) has been used in traditional medicine to treat convulsion and microbial or viral infections.Objective: In the present study, we evaluate antioxidant, antihemolytic, and protective effects of flavonoid-rich fractions of endemic Primula heterochroma Stapf. (Primulaceae) against Fe2+-induced lipid peroxidation and oxidative stress in rat brain in vitro.Materials and methods: Aerial parts of plant were defatted and extracted with 60% acetone. Then, 60% acetone extract was fractionated sequentially with n-hexane, ethyl acetate, and water. Antioxidant activity of fractions was evaluated by employing six different assays, i.e., 1,1-diphenyl-2-picryl hydrazyl (DPPH) and hydrogen peroxide scavenging, metal chelating and reducing power activities and hemoglobin-induced linoleic acid system and Fe2+-induced lipid peroxidation and oxidative stress in rat brain. Also, its antihemolytic activity was determined using 2,2′-azobis(2-amidinopropane) dihydrochloride-induced hemolysis in rat erythrocyts.Results: Among the flavonoid-rich fractions of Primula heterochroma, aqueous fraction demonstrated the most protective effect through decreasing brain thiobarbituric acid reactive substances (TBARS) levels at a dose 200 μg mL−1 (40%, p < 0.001 versus iron group). Also, the aqueous fraction showed better activity in Fe2+ chelating (89 ± 3.8 μg mL−1) and DPPH radical scavenging (394.4 ± 18.4 μg mL−1) models than other fractions. The probable protective mechanism of flavonoid-rich fractions may be attributed to their Fe2+ chelating, DPPH radical scavenging and reducing power activities. Also, the n-hexane fraction demonstrated a higher protective effect in the hemoglobin-induced linoleic acid system and 2,2′-azobis(2-amidinopropane) dihydrochloride-induced hemolysis (67 ± 2.6 μg mL−1).Discussion and conclusion: Results of this study demonstrate Primula heterochroma is a rich source of natural antioxidant compounds.