Actinidin is a cysteine protease abundant in Kiwifruit. This enzyme is known as a meat-tenderizing protease. In this project, actinidin was purified from kiwifruit by salt precipitation and ion exchange chromatography. Collagenolytic effect of the purified enzyme was tested in four different buffer systems. Thereafter, the enzyme was used for isolation and culture of cells from three different tissues: endothelial cells from human umbilical vein, hepatocytes from rat liver, and thymic epithelial cells from rat thymus. Our results revealed that actinidin can hydrolyze collagen types I and II at neutral and alkaline buffers. Furthermore, actinidin compared with type II or IV collagenase isolated intact human umbilical vein endothelial cells, hepatocytes, and thymic epithelial cells with viability more than 90%. These results address a novel and valuable collagenase, which can be used efficiently for hydrolysis of collagen and isolation of different cell populations from various solid tissues.