Abstract: Salicylic acid (SA) treatment can induce significant H₂O₂ burst in Salvia miltiorrhiza suspension culture cell and its medium alkalinization. In this study, the correlation between SA-induced H₂O₂ burst and medium alkalinization was investigated through NADPH oxidase inhibitor imidazole (IMD), H₂O₂ quenching agent dimethylthiourea (DMTU), plasma membrane H⁺-ATPase inhibitor Na₃VO₄ and plasma membrane H⁺-ATPase activator fusicoccin (FC). Results showed that (1) H₂O₂ treatment also induced significant medium alkalinization, and IMD and DMTU suppressed the process of SA-induced medium alkalinization, which suggested that H₂O₂ was involved in the process of SA-induced medium alkalinization; (2) SA suppressed the activity of plasma membrane H⁺-ATPase, Na₃VO₄ treatment caused rapid medium alkalinization and led to H₂O₂ burst ahead of time, and FC reversed SA-induced medium alkalinization and H₂O₂ accumulation, which suggested that plasma membrane H⁺-ATPase regulated the change in medium pH value and medium alkalinization promoted the generation of H₂O₂. Overall, the H₂O₂ level in the S. miltiorrhiza suspension culture cell and its medium alkalinization level functioned together to respond to induction of salicylic acid.