Objective: Proteolytic enzymes, specially collagenase, are used to digest exteracellular matrix, cells isolation and primary culture. It is important to find new sources of plant or animal protease instead of bacterial or tissue collagenase. In the present research, actinidin, a plentiful protease in kiwifruit (Actinidin deliciosa), was used for isolation and culture of cells from thymic epithelial cells (TEC) from rat thymus. Materials and Methods: Materials and Methods: The thymus was taken out. The gland was minced into small pieces and suspended in the PBS containing 1, 2, 4, 8, or 16 mg/ml actinidin for 1, 2, 3, or 4 h with gentle shaking. The cell pellet was resuspended in William’s E culture medium. The cell suspensions were cultured in dishes precoated with collagen. Results: Rat TEC was properly isolated after digestion of thymus in 4 mg/ml actinidin for 4 h at 37 °C. The isolated cells were adhered to collagen precoated dishes after washing. After 24 h of culture, the adherent cells were flattened and showed polygonal morphology with small nuclei. The viability of the cells as judged by the trypan blue test was estimated to be 90–95% in all isolations. Conclusion: The results showed that actinidin has not toxic effect on separated cells and is a novel and suitable protease for isolation of rat TEC.