Abstract: The purpose of this study was to investigate the effects of dietary energy source structure on serum biochemical indices, small intestinal tissue morphology, barrier function and cecal flora structure in mice. Seventy-two healthy 21-day-old male C57BL/6J mice with similar initial body weight were randomly divided into CON group（79% waxy corn starch）, LSB group（70% waxy corn starch +9% soybean oil）, MSB group（61% waxy corn starch +18% soybean oil） and HSB group（52% waxy corn starch +27% soybean oil）, with 18 animals in each group, which were fed four kinds of isoenergetic and isonitrogen diets（waxy corn starch and soybean oil provided all energy except quantitative protein）. The experiment lasted for 36 days. Growth performance, serum biochemical indices, intestinal mucosal morphology, barrier function and cecal microflora structure of mice were measured to study the influence of dietary energy source structure. The results showed that compared with the CON group, the final body weight of mice in MSB group and HSB group was increased by 5.61% and 7.69%, respectively（P＜0.05）. Compared with CON group, serum ALB content in LSB group, MSB group and HSB group was significantly decreased（P＜0.05）. Serum BUN content in HSB group was significantly increased by 20.11%, which was a significant difference（P＜0.05）. The TP content of LSB group and MSB group was decreased by 4.10% and 3.82%, respectively without significant difference（P＜0.05）. Compared with CON group, the depth of duodenal recess in MSB group was significantly decreased（P＜0.05）. The mRNA relative expression of Claudin-1 in the ileum of LSB group was significantly decreased compared with the other three groups（P＜0.05）. The mRNA relative expression of atresia connexin-1（ZO-1） in duodenum and jejunum of mice in HSB group was significantly increased compared with that in LSB group（P＜0.05）. In terms of the structure of cecum flora, the relative abundance of Firmicutes/Bacteroidetes decreased first and then increased; compared with CON group, the relative abundance of Firmicutes/Bacteroidetes was significantly decreased in LSB and MSB groups（P＜0.05）, while it was increased in HSB group but not significantly（P＞0.05）. At the genus level, the relative abundance of Brautella, Trichospirillum and Tremillum in MSB and HSB groups was significantly increased（P＜0.05）. These results indicated that proper adjustment of the energy source structure in the diet could promote the growth of mice, affect the serum biochemical indexes of mice, improve the intestinal barrier function, the intestinal mucosal morphology, and the relative abundance of acid bacteria in the cecum of mice, thus enhancing the mechanical barrier effect of the intestinal mucosa.