Antioxidant system is of great importance for organisms to regulate the level of excessive reactive oxygen species (ROS) under the environmental stresses including antibiotics stress. Effects of norfloxacin (NOR) on cystathionine-β-synthase (CBS), nitric oxide synthase (NOS) and antioxidant enzymes were investigated, and interaction between NO and HS and their regulation on the antioxidant system of under NOR were determined as well in the present study. After treated with 2 µg/mL NOR (1/2 MIC), CBS content, HS and NO contents decreased while HO accumulation and the antioxidant-related genes mRNA level increased. Additionally, the endogenous HS content in was increased by the exogenous NO, while HO accumulation and the relative expression level of (Superoxide dismutase gene) decreased under exogenous NO or HS. And the content of endogenous NO and NOS in increased under the exogenous HS as well. Taken together, these results showed that anti-oxidative ability in was respectively enhanced by the gas molecules of HS and NO under NOR-induced stress, and there may be a crosstalk regulative mechanism between HS and NO. These results lay a foundation for the research of regulation network of HS and NO, and provide a hint to synthesize anti-vibrio drugs in the future.