Purpose The aim of the present study was to investigate the efficacy of recombinant human endostatin (ES) (rh-ES) combined with radiation on rat cardiomyocyte apoptosis and the regulatory mechanism of transforming growth factor beta1 (TGF-beta1)/Sma and Mad-related protein 3 (Smad3)/connective tissue growth factor (CTGF) signaling. Method The primary cardiomyocytes were isolated from neonatal Sprague-Dawley rats for culture in vitro and divided into blank control group (without treatment), 10 Gy radiation + siTGF-beta1 siRNA (gene silencing) group, ES + siTGF-beta1 siRNA group, and 10 Gy radiation + ES + siTGF-beta1 siRNA group. Methyl thiazolyl tetrazolium assay was used to calculate the half-maximal inhibitory concentration (IC.sub.50) of rh-ES on cardiomyocytes. Adenoviral vector was constructed for virus packaging to silence TGF-beta1 expression in cardiomyocytes. Quantitative real-time polymerase chain reaction and Western blot were carried out to analyze TGF-beta1, Smad2, Smad3 and CTGF expression at both gene and protein levels. Flow cytometry and electron microscope were used to examine cell apoptosis. Results ES had a dose-dependent inhibitory effect on the proliferation of primary rat cardiomyocytes. ES combined with radiotherapy significantly inhibited cardiomyocyte proliferation and promoted cell apoptosis (P < 0.01). The gene and protein expression of TGF-beta1, Smad2, Smad3 and CTGF were significantly up-regulated in primary cardiomyocytes transfected with TGF-beta1 gene (P < 0.05). Conclusion The combination therapy with rh-ES and radiation can promote cardiomyocyte apoptosis and aggravate myocardial cell damage via TGF-beta1/Smad3/CTGF signaling pathway. Keywords: Recombinant human endostatin, Radiotherapy, Signaling pathway, Apoptosis, Lung cancer