Abstract: Objective To investigate effects of different Cryopreservation time on functional and the activity changes of NK cells. Methods To collect and isolate single nuclear cells in peripheral blood of healthy peple, and in vitro amplification and culture of NK cells , collect and frozee some NK cells in -80℃ after 12 days , Frozen storage time: 1 week, 4 weeks, 12 weeks and 24 weeks. The expression and comparison of the surface markers, apoptosis percentage, Perforin, Granzyme B and CD107a were detected by flow cytometry;LDH method was used to detect the killing activity of NK cells before and after freezing and compared with those of esophageal cancer cell lines. Results there was no significant change in the surface markers、apoptosis、Perforin、Granzyme B 、CD107a and the killing activity of esophageal cancer cell lines of the cells at 1 week, 4 weeks and 12 weeks; but there were significant differences between the test group and then on-frozen storage group at 24 weeks（P<0.05）. Conclusions There was no significant change on cell function and the killing activity of esophageal cancer cell lines before 12 weeks -80℃ cryopreserved ; but there were significant differences after 24 weeks -80℃ cryopreserved