Objective To investigate the feasibility of recombinant lentivirus (LVs) mediated hyperpolarization- activated cyclic nucleotide-gated cation channel 4 (HCN4) gene transfecting rat bone mesenchymal stem cells (BMSCs) so as to construct the biological pacemaker cells. Methods Sprague Dawley rats at the age of 3-5 weeks were selected to isolate and culture BMSCs using modified whole bone marrow adherent culture method. LVs was used as carrier, and enhanced green fluorescent protein (EGFP) as marker to build LVs-HCN4-EGFP virus liquid. The BMSCs at passage 3 were transfected with LVs-HCN4-EGFP virus liquid (experimental group) and LVs-EGFP null virus liquid (control group). Fluorescence microscope was used to observe the green fluorescent protein expression after 24, 48, and 72 hours of transfection; Western blot method was used to detect the HCN4 protein expression. The electrophysiology was used to detect the pacemaker current in the experimental group. Results After transfection, BMSCs in the experimental group showed normal morphology and good growth; scattered green fluorescence could be seen at 48 hours under fluorescence microscope, with a transfection efficiency of about 10%; the fluorescence expression increased slightly, with the transfection efficiency of 20% to 25% at 72 hours. While no expression of green fluorescence was seen in the control group. Western blot results showed that the same band expression as a relative molecular mass of HCN4 protein were found at 72 hours after transfection in the experimental group, only weak expression of protein band was seen in the control group; the gray value of the experimental group (33.75 ± 0.41) was significantly higher than that of the control group (23.39 ± 0.33) (t=17.524, P=0.013). In the experimental group, the pacemaker current was recorded, and it could be blocked by CsCl, in accordance with the characteristics of pacemaker current. Conclusion The recombinant LVs mediated HCN4 gene is successfully transfected into rat BMSCs, and the expression of HCN4 protein and the pacemaker current can be detected.