Abstract: AIM To establish an efficient method for culturing human atrial fibroblasts in vitro. METHODS Samples of human right atrial tissue were cut into 1 mm3 pieces by aseptic technique. Then, these pieces were cultured using explants adherent method directly after predigestion treatment by 1.25 g/L trypsin for 15 minutes. The morphology and growth of the culture cells were observed under an inverted microscope and the fibroblastic specificity was identified by immunofluorescence cell staining. RESULTS The cells migrated from the explants in 3-4 days and with first migration identified in 7-9 days. The migrated cells were long spindle or polygon-shaped and had a positive reaction to antibodies against vimentin and α-SMA and a negative reaction to antibodies against vWF for immunofluorescence staining. They were identified as myofibroblasts. CONCLUSION The technique of tissue explant with enzymatic digestion is a convenient, efficient, and feasible method for the culturing of human atrial fibroblasts/myofibroblasts in vitro.