Objective To establish a method for isolation, culture and identification of primary human pericardial interstitial cells (PICs). Methods Human pericardial tissues were cut into 1 mm×1 mm×1 mm sized pieces, digested with type Ⅱ collagenase, hyaluronic acid enzyme and trypsin to isolate the interstitial cells. The cells were cultured and cell morphology was observed. The cell doubling time, flow cytometry and immunohistochemical staining were applied for cell identification. Results Adherent cells were observed 24 h after culturing. The cells reached 80% confluences and could be subcultured after about 10 days. The cells isolated and cultured were fibroblast-like or spindle-like, with obvious nuclei, clear nucleolus, and a greater proportion of nucleus and cytoplasm. Flow cytometry results showed no cell surface markers CD34 and CD45. Immunocytochemistry showed negative CK staining, and positive vimentin and α-SMA staining. Cells isolated from 8 of the 10 pericardial tissues were successfully cultured. Conclusion The present method can effectively isolate and culture primary human PICs, which lays a foundation for further in vitro study of constrictive pericarditis.