Feeding habit determines the digestive tract structure and intestinal microflora. However, the relationship among feeding habit, digestive physiology intestinal, and microbial diversity of omnivorous, herbivorous, filter-feeder and carnivorous fish reared in the same pond has not been compared. This study compared the digestive enzyme activities, intestinal morphology and intestinal microflora of omnivorous (Carassius auratus), herbivorous (Ctenopharyngodon idellus), carnivorous (Siniperca chuatsi) and filter-feeder (Shizothorax grahami) and predicted the potential functions of specific microflora on different nutrients. Twelve intestine samples were collected from each of the four fishes from Dianchi Lake. The composition and diversity of microbial communities were determined by using high throughput sequencing of 16S rDNA. The results showed that the filter-feeder fish had significantly higher protease but lower amylase activities in the intestine than herbivorous. The carnivorous fish intestine had more microvilli branches and complex structures than other fish species in the order carnivorous > herbivorous > filter-feeder > omnivorous. The diversity of intestinal microflora was higher in omnivorous and followed the order omnivorous > herbivorous > filter-feeder > carnivorous. Acinetobacter species and Bacteroides species were the most dominant flora in carnivorous and herbivorous fish, respectively. Acinetobacter johnsonii, Acinetobacter lwoffii and Pseudomonas stutzeri might help the host to digest protein, while Bacteroidetes species may help the host to digest cellulose. Taken together, feeding habit determines the digestive enzyme activities, intestinal tissue morphology and differential colonization of fish intestinal flora. The knowledge obtained is useful in designing appropriate approaches for feed formulation and feeding practices in for fish.