This study investigated the changes in half lethal low temperature (LT50), the contents of flavonoid, non-structural carbohydrates and plant hormone, as well as the proteomics on the current-year branches of Rosa beggeriana during overwintering. The results showed that the freezing tolerance of the branches gradually increased and its LT50 value reached -54.7℃ in the coldest phase of the winter. No significance on the contents of starch and ABA were observed, whereas the contents of flavonoids, total soluble sugars including sucrose, glucose, fructose and raffinose were found to be significantly increased in the branches. However, SA contents in the branches decreased at the beginning phase of the winter, followed by an increase significantly in the coldest phase of the winter. The correlative analysis showed that the LT50 values were negatively correlated with the contents of flavonoids, total soluble sugars, glucose and fructose. The proteomic analysis showed that there were total 153 differentially expressed proteins (DEPs) accumulated in branches during overwintering. Of them, there were 92 DEPs at the beginning phase of the winter and 91 DEPs in the coldest phase of the winter. The DEPs, which are annotated to involve in starch and sucrose metabolism, flavonoids biosynthesis, antioxidant defenses, cell wall relaxation and extension, water transport channels as well as cell dehydration responses were significantly up-regulated, suggesting that the branches activated a series of physiological and biochemical responses to improve the long-term freezing tolerance. Moreover, the DEPs such as dehydrin, anti-freezing protein and aquaporin may play the important roles in the cell water balances of the branches in the coldest phase of the winter.