Hydrogen sulfide (H₂S), a gasotransmitter, plays a variety of roles in the mammalian body including the cardiovascular system. Given evidence that H₂S donors including NaHS inhibit human platelet aggregation, we examined and characterized the effects of NaHS on rabbit platelet aggregation and cytosolic Ca²⁺ mobilization. Rabbit platelet aggregation was determined in platelet-rich plasma (PRP) and washed platelets. Intracellular Ca²⁺ levels were monitored in Fura2-loaded washed platelets. NaHS prevented rabbit platelet aggregation induced by collagen or ADP, and the effective concentration range of NaHS was 0.1–0.3 m M in PRP and 1–3 m M in washed platelets. In washed platelets, NaHS attenuated cytosolic Ca²⁺ mobilization induced by collagen or ADP and also reduced platelet aggregation induced by ionomycin, a Ca²⁺ ionophore. The anti-platelet effect of NaHS was blocked by an adenylyl cyclase inhibitor and enhanced by a phosphodiesterase inhibitor. H₂S thus suppresses rabbit platelet aggregation by interfering with both upstream and downstream signals of cytosolic Ca²⁺ mobilization in a cAMP-dependent manner.