Aim To construct lentiviral vector carrying the connexin43 (Cx43) gene, and make it express in the rat bone mesenchymal stem cells (BMSC). Methods The cDNA of human Cx43 gene was obtained with reverse transcription polymerase chain reaction (RT-PCR). The Cx43 gene was recombined to construct the transfer plasmid FUGW-Cx43 by infusion technique. The three-plasmid system of lentiviral vector was consisted of FUGW, the packaging plasmid, and the envelope plasmid VSVG, which were cotransfected to 293T cells mediated by lipofectamin 2000 to produce lentiviral particles. The rat BMSC were infected by obtained lentiviral particles. The expression of Cx43 protein was observed with fluorescent microscope. Results The result of sequencing showed that the cloned Cx43 gene was consistent with the sequence reported in GeneBank. The insertion of Cx43 gene in viral genome was confirmed by PCR. The FUGW-Cx43 plasmid was identified to have correct sequence. After the three plasmids of lentiviral vectors were cotransfected to the 293T cells, the supernatant was collected and concentrated, the titer of the lentiviral vector particles was found to be 1×1011 TU/L. Cx43 gene-modified rat BMSC show linear and stippled patterns of Cx43 expression on cell membrane. Conclusion Lentiviral vector carrying Cx43 gene has been successfully constructed. The infected rat BMSC are able to express the Cx43 protein. This will facillitate the following exploratory development of Cx43 gene modified stem cell-based cardiac repair.