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Objective: This study aimed to reconstruct histidine-tagged human large conductance Ca2+-sensitive K+channels (BKCa) in HEK293 cells.Methods: BKCa DNA was cloned using reverse transcription PCR from human mesenteric artery and connected with pEF1/HisB vector.BK-pEF1/HisB recombinant plasmid was transfected into HEK293 cells using liposome transfection method.The expression of BKCa was detected with western blot and patch clamp technique.Results: BKCa DNA which we cloned from human mesenteric artery contained 3339 bp nucleotides and could encode 1113 amino acids.It was inserted into pEFl/HisB vector between Kpn I and Bam HI endonuelease sites with correct open read fragment.According to our design, a 48 amino acid histidine tag was added in the C-terminal of the target BKCa protein.It was helpful for us to purify BKCa protein in our future research.There was BKCa protein expression in HEK239 cells after transient transfection.BKCa single channel current could be recorded in this human BKCa HEK293 cells and the characteristics of single channel including large conductance, voltage dependence and calcium sensitivity were the same as those found in nature human mesenteric artery smooth muscle cells.Conclusion: BKCa gene was cloned from human mesenteric artery and a functional BKCa ion channel was constructed in HEK293 cells.