Alfalfa often suffers from low temperature during spring rejuvenation, so it is important to improve the cold tolerance of alfalfa leaves for its smooth rejuvenation, and the alternative pathway (AP) could effectively improve the plant's tolerance. In this study, the contribution of AP on spring rejuvenation of alfalfa was investigated in Xinmu No.4 and Gannong No.5 with different fall dormancy levels. Though the protein and AP capacity were decreased during the rejuvenation, the ratio of AP/TP were increased in two alfalfa varieties, compared to those in alfalfa before overwintering. This indicated that AP had positive response to alfalfa rejuvenation. The limit ation of AP significantly affected the leaf length, leaf width and growth rate of greening alfalfa, showing that AP played an important role in alfalfa rejuvenation. Inhibition of AP resulted in a significant decrease in Pn, Ci, Gs and stomatal structure deformity, suggestting that AP affected photosynthesis by influencing stomatal development during rejuvenation. AP reduces oxidative damage to PSII core protein repair in alfalfa leaves and optimizes photosynthesis by up-regulating NADP-MDH activity, decreasing the accumulation of excess reducing power in the chloroplasts, and by increasing SOD and POD activities and decreasing the accumulation of hydrogen peroxide. The higher proportion of AP keeps it more tolerant to low temperature for rejuvenation in Xinmu No.4 with a lower fall dormancy level.