Background: Atelocollagen, a biocompatible natural polymer, has been used successfully to deliver genetic materials into cells. Methods: The nanoparticles containing reporter genes encoding firefly luciferase (Fluc) or secreted alkaline phosphatase (SEAP) were embedded on atelocollagen coated culture plate. A commercial lipid-based transfection reagent (reagent X) was used to compare the efficacy of transfection. Cardiomyocytes were isolated from neonatal rat and seeded on the coated culture plate. The expression rate and kinetics were confirmed by assays of expressed luciferase or SEAP. Cell toxicity was assessed by WST-1 assay. To confirm the transfection of functional gene, IL-10 gene was cloned into gWIZ eukaryotic expression vector. After transfection of IL-10 into cardiomyocytes, RT-PCR and ELISA were performed to confirm IL-10 mRNA and protein release, respectively. Results and Conclusions: Two days after cell seeding on polyplex-immobilized surface, cell viability and gene expression were evaluated. The cell viability was almost 100% in atelocollagen group, whereas 79% in reagent X group in 2 days. The luciferase activity was increased to 80,000-fold and SEAP activity was increased to 2.4-fold in atelocollagen group. The SEAP activity was increased to 2.1-fold in reagent X group. Taken together, the nanoparticles, formed from the complex of DNA and positively charged polyethylenimine coated on collagen layer, were efficiently transduced into cardiomyocytes without a significant cytotoxicity. As a therapeutic gene, IL-10 was chosen because IL-10 is an anti-inflammatory cytokine and is expected to be utilized to protect cardiomyocytes from inflammation. After transfection of IL-10 to cardiomyocyte, IL-10 mRNA was significantly increased in comparison with non-transfected cardiomyocytes and the level of released IL-10 protein was also increased to 25.85 pg/mL (4105 cardiomyocytes) without any noticeable cytotoxicity. These results suggest that this novel technique using optimized reverse transfection approaches may be appropriate for ex vivo gene therapy designed to produce genetically controlled cardiomyocytes for cell therapy.