〔Abstract〕 Objective To investigate the role of connexin 43(Cx43) in acute sympathetic stress induced by phenylephrine(PE) overactivation of α1-adrenergic receptor(α1-AR) in cardiomyocytes. Methods Cardiomyocytes of H9 C2 rats were randomly divided into control group, PE alone treatment group, Gap26(Cx43 specific inhibitor) intervention group and Gap26 alone treatment group. PE alone treatment group was treated with 50 μmol/L PE for 15 min. The Gap26 intervention group was pretreated with 0.5 μmol/L Gap26 for 30 min, and then treated with 50 μmol/L PE for 15 min. The protein and mRNA expression levels of Cx43, NLRP3 inflammasome, interleukin-1β(IL-1β), Caspase-1, interleukin-18(IL-18) were detected by Western blot and qRT-PCR. The expression and co-location of Cx43 in cardiomyocytes were observed by immunofluorescence assay, and the expression of inflammatory cytokines IL-1β and IL-18 in cardiomyocytes was detected by ELISA. Results Compared with control group, the protein and mRNA levels of Cx43, NLRP3, Caspase-1 and IL-18 in PE group increased. Compared with PE alone treatment group, the protein and mRNA levels of Cx43, NLRP3, Caspase-1 and IL-18 decreased after Gap26 intervention, but they were still higher than those of control group. Similarly, immunofluorescence showed that Cx43 protein expression increased in PE alone group, while Cx43 expression was down-regulated in Gap26 intervention group compared with PE alone group. ELISA results showed that the expression of IL-1β and IL-18 was significantly up-regulated in PE alone group, but down-regulated in Gap26 intervention group. Conclusion Cx43 is involved in α1-AR activation induced cardiac acute sympathetic stress by regulating NLRP3 inflammasome.