Biotin is a member of the vitamin B-complex family. Biotin deficiency has been associated with hyperglycaemia and insulin resistance in animals and humans. In the present study, we investigated the pharmacological effects of biotin on hypertension in the stroke-prone spontaneously hypertensive rat (SHRSP) strain. We observed that long-term administration of biotin decreased systolic blood pressure in the SHRSP strain; also, a single dose of biotin immediately decreased systolic blood pressure in this strain. Pretreatment with the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazole [4,3-α]quinoxalin-1-one abolished the hypotensive action of biotin in the SHRSP strain, while pretreatment with the NO synthase inhibitor NG-nitro-l-arginine methyl ester had no effect on the action of biotin. Biotin reduced coronary arterial thickening and the incidence of stroke in the SHRSP strain. These results suggest that the pharmacological dose of biotin decreased the blood pressure of the SHRSP via an NO-independent direct activation of soluble guanylate cyclase. Our findings reveal the beneficial effects of biotin on hypertension and the incidence of stroke.