Aim To explore the effect of the blocker of gap junction on phenotypic transition in cultured rat vascular smooth muscle cells. Methods Rat aortic smooth muscle cells (SMC) were cultured by explanted rat aortic wall tissue. Cell immunofluorescence staining was applied to detect the expressions of connexin (Cx)43 in rat aortic SMC. Fluorescence redistribution after photobleaching (FRAP) was used to measure the communications between cells via gap junctions. MTT and RT-PCR were hired to measure the proliferative capability of rat aortic SMC and the expression of smooth muscle (SM) α-actin respectively. Meanwhile, 18α-glycyrrhetinic acid (18α-GA), a specific blocker of gap junction, was administered to observe its effect on the contents above. Results At 3rd day after cultured, Cx43 was expressed in rat aortic SMC. Fluorescent dye could only be transferred between conjugated cells, and mean fluorescence recovery rate in isolated cells were significantly lower compared with that in conjugated cells (7.30%±0.58% vs 80.61%±6.57%, P<0.01). Compared to control group, mean fluorescence recovery rate in 18α-GA group were significant lower (61.43%±7.62% vs 80.61%±6.57%, P<0.05). Therefore, 18α-GA could inhibit dye transfer between conjugated cells. 18α-GA could also inhibit the proliferation of rat aortic SMC and promote the expression of SM α-actin in cultured rat aortic SMC. Conclusions The blocker of gap junction could promote the phenotypic transition rat aortic SMC from the synthetic to the contractile phenotype, which suggested that gap junction played a role on the phenotypic modulation in cultured rat aortic MSC.