The experiment was conducted to study the effects of different overwintering feeds on midgut digestive enzyme activities, tissue development status and antioxidant enzyme gene expression of honeybees, in order to provide reference for the beekeepers choose winter feed overwintering feeds. At the end of October 2015, nine overwintering colonies (without overwintering feed reserve) of local Italian honeybees (Apis mellifera L.) with equal colony population were randomly divided into 3 experimental groups (3 colonies per group), and were fed honey (honey group), fructose corn syrup (fructose corn syrup group) and white sugar water (white sugar:water=2:1, sucrose group) as overwintering feed from November 2 to late November when bee colonies entered into overwintering period, respectively. The experiment lasted from the end of October 2015 to early March 2016. In order to determine the midgut digestive enzyme (amylase, sucrase and protease) activities, midgut samples were collected in early November (before overwintering), early January (the middle period of overwintering) and early March (after overwintering), respectively. And, in early January, midgut samples were collected to determine the midgut tissue development status and the relative expression levels of antioxidant enzyme genes, such as superoxide dismutase 1 (Sod1), superoxide dismutase 2 (Sod2) and catalase (CAT) genes. The results showed as follows:the midgut amylase activity of fructose corn syrup group and honey group was significantly higher than that of sucrose group in early January (P<0.05). Different overwintering feeds did not significantly affect the midgut sucrase activity in early January and early March (P>0.05). The midgut protease activity of fructose corn syrup group was significantly higher than that of sucrose group and honey group in early January (P<0.05). The midgut wall thickness of sucrose group and honey group was significantly higher than that of fructose corn syrup group (P<0.01), and the midgut crypt deepness of sucrose group and honey group was also significantly higher than that of fructose corn syrup group (P<0.01). The relative expression level of Sod1 gene from midgut of sucrose group was significantly higher than that of fructose corn syrup group and honey group in early January (P<0.05). In conclusion, honey can improve midgut digestive enzyme activities of overwintering honeybees, honey and white sugar are beneficial to the development of midgut tissue of overwintering honeybees, and white sugar can improve the midgut antioxidant gene expression of overwintering honeybee. Therefore, honey and white sugar as the overwintering feed for honeybee are better than fructose corn syrup.