We investigated the changes in the contents of H2O2, malonaldehyde (MDA) and endogenous antioxidants, the activities of protective enzymes and some critical enzymes involved in the ascorbate-glutathione (ASA-GSH) cycle as well as freezing resistance(expressed as LT50) and correlations mentioned above, in detail using Populus suaveolens cuttings. The purpose was to explore the physiological mechanism of the enhancement of freezing resistance induced by freezing acclimation at -20℃, and to elucidate the physiological mechanisms by which trees adapt to freezing. The results showed that freezing acclimation enhanced the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), monodehydroascorbate reductase (MDAR), ascorbate peroxidase(APX), dehydroascorbate reductase (DHAR) and glutathione reductase (GR). And it increased the contents of reduced ascorbate(ASA), reduced glutathione (GSH), dehydroascorbate (DHA) and oxidized glutathione (GSSG). However, H2O2 and MDA contents and LT50 of cuttings were decreased. LT50 in cuttings was found to be closely correlated to the levels of SOD, POD, CAT, APX,DHAR, MDAR, GR, H2O2, MDA, ASA, GSH, DHA and GSSG during freezing acclimation. This suggested that the enhancement of freezing resistance of cuttings induced by freezing acclimation may relate to the distinct increase for the levels of SOD, POD, CAT,APX, DHAR, MDAR,GR,ASA, GSH, DHA, and GSSG. In addition, the observed levels of APX, DHAR, MDAR, GR, ASA, DHA,GSH and GSSG were higher than those of SOD, POD and CAT during freezing acclimation. It indicated that a higher capacity of the ASA-GSH cycle is required for H2O2 detoxification, and growth and development of cuttings. Based on the obtained results, it can be concluded that the ASA-GSH cycle plays an important role in enhancement of freezing resistance of P. suaveolens cuttings during freezing acclimation.