Objective To construct models of viral transfection and hypoxia / reoxygenation induced cellular injury in adult mouse cardiomyocytes (AMCMs) isolated using a non-Langendorff method. Methods AMCMs were isolated,extracted, sedimented, and plated using a non-Langendorff method. The morphology and survival rate of the isolated cells were evaluated 2, 24, 48 and 72 h after plating, and their integrity was observed by immunofluorescence staining for αactinin. The isolated AMCMs were infected with adenoviruses carrying an RFP-expressing vector and fluorescence images were obtained at 36 and 48 h post-infection and used to calculate transfection efficiency. The cells were cultured under hypoxic conditions for 45 min, reoxygenated for 24 h, and then stained with propidium iodide (PI) to verify establishment of the hypoxia / reoxygenation injury model. Results The survival rates of AMCMs at 2, 24 and 48 h after plating were comparable, but survival was significantly reduced at 72 h. The integrity of the AMCMs was good and > 80% of the cells were transfected with adenovirus at 48 h. After hypoxia / reoxygenation treatment, 42% of cells were stained by PI, suggesting successful establishment of the AMCM injury model. Conclusions In this study, we developed a nonLangendorff method for the fast and easy isolation of AMCMs with high cell viability. The isolated cells can be efficiently infected with adenovirus and respond to hypoxia / reoxygenation injury. These findings provide a systematic method for isolating AMCMs and for applying gene modification and hypoxia / reoxygenation injury in these cells.