Objective To explore the isolating methods of rat submandibular gland cell for primary culture. Methods Rat submandibular gland cell were isolated by direct isolation and pancreatin digestion respectively, and then were cultured and subcultured on DMEM. The shape and structure of cultured cells were observed with phase contrast microscope. The cell survival rate was detected by using trypan blue elimination test. The vital force of culture cells was estimated with MTT colorimetric method. The cultured cell secretion function was evaluated by assay of amylase activity. Results By direct isolatin, the cell survival rate was 70% and the cell vital force was 0.16±0.014. By pancreatin digestion, the cell survival rate was 85% and the cell vital force was 0.45±0.13; the cells had good shape and attached well. The Ck8.13 and keratin antibodies were epithelium specific and α-SMA antibodies were myoepithelium specific. The cells were stained positively with CK8.13, keratin and α-SMA antibodies. Conclusion The method of pancreatin digestion for the isolation of submandibular gland cell is better than that of idrect isolation.