Abstract： Objective To investigate the effect of substance P (SP) on expression of β1-adrenergic receptor (β1-AR) in cultured neonatal rat cardiomyocytes. Methods Cardiomyocytes were obtained from 1- 3 days old Sprague-Dawley (SD) rats and cultured for 72 hours. Six wells of the cultured cells were divided into two groups (n= 3 in each group): control group and SP group (incubated with 10-7 mol/L of SP). Immunocytochemistry was used to detect the expression of β1-AR in cultured rat cardiomyocytes. Fifteen wells of the cultured cells were divided into five groups (n=3 in each group): control group and four SP groups (incubated with 10-9, 10-8, 10-7, 10-6 mol/L of SP respectively). Quantitative analysis of the expression of β1-AR in cultured neonatal rat cardiomyocytes of all the groups was performed using flow cytometry. Results The results of immunocytochemistry showed that the expression of β1-AR in cardiomyocytes in SP group was lower than that in control group (positive unit: 179.16 ± 48. 18 vs. 205.79±117. 42, average light density: 128. 26±22. 72 vs. 157.35±33. 11, both P<0. 05). The results of flow cytometry showed that significantly lower levels of expression of β1-AR were observed in the groups treated with SP in the concentrations ranging from 10-8 mol/L to 10-6 mol/L (151.85± 4.63, 135.00±6.84, 121.41±5.22 vs. 161.35±3.09, all P<0. 05). Conclusion SP could down-regulate the expression of β1-AR in cultured rat cardiomyocytes in vitro.