This study was designed to investigate the cardiovascular effects of sulfur dioxide (SO2) in the caudal ventrolateral medulla (CVLM) of anesthetized rats and its mechanism. Different doses of SO2 (2, 20, 200 pmol) or artificial cerebrospinal fluid (aCSF) were injected into the CVLM unilaterally or bilaterally, and the effects of SO2 on blood pressure and heart rate of rats were observed. In order to explore the possible mechanisms of SO2 in the CVLM, different signal pathway blockers were injected into the CVLM before the treatment with SO2 (20 pmol). The results showed that unilateral or bilateral microinjection of SO2 reduced blood pressure and heart rate in a dose-dependent manner (P < 0.01). Moreover, compared with unilateral injection of SO2 (2 pmol), bilateral injection of 2 pmol SO2 produced a greater reduction in blood pressure. Local pre-injection of the glutamate receptor blocker kynurenic acid (Kyn, 5 nmol) or soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1 pmol) into the CVLM attenuated the inhibitory effects of SO2 on both blood pressure and heart rate. However, local pre-injection of nitric oxide synthase (NOS) inhibitor NG-Nitro-L-arginine methyl ester (L-NAME, 10 nmol) only attenuated the inhibitory effect of SO2 on heart rate but not blood pressure. In conclusion, SO2 in rat CVLM has cardiovascular inhibitory effects, and its mechanism is related to the glutamate receptor and NOS/cGMP signal pathways.