Large conductance Ca2+-activated K+ (BKCa) channel exhibits a phenotype-dependent expression on vascular smooth muscle cells (VSMCs), which prefers to contractile phenotype. Meanwhile, shear stress definitely influences VSMCs proliferation and contraction. Thereby, a hypothesis was raised, would shear stress change the BKCa expression and correlate with VSMC phenotype? In order to investigate it, VSMCs were exposed to shear stress in a parallel-plate flow chamber with 12 dynes/cm2 for 12 h. Subsequently, the effect of shear stress on VSMC proliferation, BKCa channel expression and contractile phenotype marker, α-smooth muscle cell actin (α-SMA) and smooth muscle myosin heavy chain (SMMHC), was determined by immunofluorescence microscopy, flow cytometery as well as reverse transcription-polymerase chain reaction (RT-PCR), respectively. Data show that shear stress enhanced the expression of BKCa channel while inhibiting VSMC proliferation. Paralleled to those phenomena, the expression of both α-SMA and SM-MHC were decreased significantly. These results demonstrated that upregulation of BKCa channel was irrelevant to the maintenance VSMC of contractile phenotype under shear stress. This finding provides a new insight into understanding the correlation of BKCa channel and VSMC phenotype.