Recent progress reveals that the damaged adult Xenopus tropicalis heart is able to be regenerated in a nearly scar-free manner in around 30 days after apical resection. It is believed that cardiac fibroblasts will paly an important role in the scar-free regeneration of adult Xenopus heart. However, the methodology for isolation and culture of adult cardiac fibroblasts in adult Xenopus heart is not yet reported. In this study, an in vitro methodology for isolating and culturing cardiac fibroblasts of adult Xenopus heart was successfully established.