Objective: To study the functional properties of I<sub>f</sub> channels and the changes in mechanical activity of mouse mesenchymal stem cells (mMSCs) transfected with mHCN4. Methods: mMSCs were purified by using CD11b-immunomagnetic microbeads and transfected with pMSCV-mHCN4-EGFP or pMSCV-EGFP. We examined the kinetic characteristics of the mHCN4 channel. The morphological changes of positively transfected mMSCs were investigated at the same time. Results: The I<sub>f</sub> current recorded from the experimental group was sensitive to extracellular Cs<sup>+</sup> (–44.5 ± 4.2 vs. –5.5 ± 1.0 pA/pF, p < 0.001). The half-maximal activation was –99.0 ± 5.8 mV. The time constant of activation was 451 ± 61 ms under –140 mV. The control cells did not show the current under the same conditions. The absolute values of half-maximal activation decreased in the presence of cAMP or cGMP in the experimental group (–78.6 ± 10.4 and –85.7 ± 8.6 vs. –99.0 ± 5.8 mV, respectively, p < 0.05). mMSCs transfected with pMSCV-mHCN4-EGFP could form spontaneous beating cells. Extracellular Cs<sup>+</sup> decreased the beating rate significantly (196 ± 50 vs. 66 ± 23 bmp, p < 0.01). Conclusions: Functional I<sub>f</sub> channels can be reconstructed in mMSCs infected with mHCN4. mMSCs modified by successful transfection with mHCN4 can differentiate so as to develop spontaneous mechanical activity.