To investigate the effects of different concentrations and different administration time of salvianolic acid B (SalB) on regulating TLR4-NFκB-TNFα inflammatory pathway in hypoxia neonatal rat cardiomyocytes. Methods:1-3 d neonatal cardiomyocytes were isolated and purified, and hypoxia model was established in vitro. SalB high, medium and low dose concentrations were 10-5 mol/L, 10-6 mol/L and 10-7 mol/L, and administered to cells at 6h before hypoxia, same time with hypoxia and 6h after hypoxia respectively. TLR4 and NFκB mRNA expression were detected by qRT-PCR; TLR4 and NFκB protein expression were detected by immunohistochemical method. The concentration of supernatant TNFα was detected by ELISA. Results:Compared with normal group, the expression of TLR4 and NFκB mRNA and protein, the concentration of TNFαin supernatant were significantly increased in hypoxia model group (P<0.05 or P<0.01). Compared with the hypoxia model group, the expression of TLR4 and NFκB mRNA and protein, the concentration of TNFα in supernatant were significantly decreased in large and middle dose of salvianolic acid B administered before and meantime of hypoxia group (P<0.05 or P<0.01). The most significant decrease was observed in the high-dose group administered before hypoxia (P<0.01).Conclusion:TLR4-NFκB-TNFα inflammatory pathway was significantly activated 6h after hypoxia injury. Salvianolic acid B administered before and same time of hypoxia protected cardiomyocytes tremendously. This effect was related to the inhibition of TLR4-NFκB-TNFα inflammatory pathway. Salvianolic acid B showed dose and time related effects, and the best effect was observed in the high-dose group administered before hypoxia.