Objective To reveal the efficiency and mechanism of C1q tumor necrosis factor-related protein 3 (CTRP3) regulating Sendai virus (SeV) vector overexpression of Gata4, Mef2c and Tbx5 (SeVGMT) to reprogram cardiac fibroblasts (CFs) into induced cardiomyocyte-like cells (iCMs). Methods CFs were divided into Control group, NC-Lv group, CTRP3-Lv group, NC-sh group and CTRP3-sh group. NC-Lv, CTRP3-Lv, NC-sh and CTRP3-sh were transfected into CFs using Lipofectamine3000 reagent for 48 h. Then Lipofectamine3000 reagent was mixed with SeVGMT and incubated at room temperature for 48 h. The culture medium was replaced and then cultured for 21 d. The cell morphology at different culture time points (0, 3, 7, 14, 21 d) was observed under a microscope. The expression of myocardial specific proteins α-MHC, α-actin, cTnT, Cx43, Actc1 and Myh6 at different time points were detected by cell immunofluorescence, qRT-PCR and Western blot, and the proportion of beating cells at different time points was calculated. Results With the extension of culture time, the relative fluorescence intensity, mRNA and protein levels of α-MHC, α-actin, cTnT, Cx43, Actc1 and Myh6 in CFs of each group increased (P<0.05), and the expression levels of the above myocardial specific proteins at 14 d of culture were significantly higher than those at 7 d of culture (P<0.05). At 3 d, 7 d, 14 d and 21 d of culture, the relative fluorescence intensity, mRNA and protein levels of α-MHC, α-actin, cTnT, Cx43, Actc1 and Myh6 in CFs of CTRP3-Lv group increased compared with those of NC-Lv group (P<0.05). Compared with the NC-sh group, the relative fluorescence intensity, mRNA and protein levels of α-MHC, α-actin, cTnT, Cx43, Actc1 and Myh6 in CFs of the CTRP3-sh group were decreased (P<0.05). At 7 d of culture, beating cells appeared in CFs of each group. With the extension of culture time, the proportion of beating cells in CFs of each group increased (P<0.05), and the proportion of beating cells at 14 d of culture was significantly higher than that at 7 d of culture (P<0.05). At 7 d, 14 d and 21 d of culture, compared with the NC-Lv group, the proportion of beating cells in CFs of the CTRP3-Lv group increased (P<0.05). Compared with the NC-sh group, the proportion of beating cells in CFs of the CTRP3-sh group decreased (P<0.05). Conclusion CTRP3 can enhance SeVGMT reprogramming of CFs into iCMs.