Simple SummaryThere have been reports on alternate feeding between diets based on different lipid sources, but how the intestinal microbiota of fish responds to alternate feeding strategies and its relevant roles in the host’s health have not been reported. The present study provides novel results in this area. The main objective of the study was to assess how the intestinal microbiota of fish responds to alternate feeding between diets based on different lipid sources. Foremost, the juvenile turbot accepted alternate feeding between diets based on different lipid sources with no negative effects on survival or growth performance. Additionally, novel results were observed regarding the different bacterial compositions, microbial co-occurrence networks, and functional predictions among the different alternating feeding strategies. The results of this study indicate that a more comprehensive evaluation should be conducted from the perspective of intestinal microorganisms when a new feeding strategy is applied in aquaculture practices. These results will contribute to the nutritional regulation of aquatic animals from the perspective of microorganisms.AbstractA nine-week feeding trial was conducted to investigate changes in the intestinal microbiota of turbot in response to alternate feeding between terrestrially sourced oil (TSO)- and fish oil (FO)-based diets. The following three feeding strategies were designed: (1) continuous feeding with the FO-based diet (FO group); (2) weekly alternate feeding between soybean oil (SO)- and FO-based diets (SO/FO group); and (3) weekly alternate feeding between beef tallow (BT)- and FO-based diets (BT/FO group). An intestinal bacterial community analysis showed that alternate feeding reshaped the intestinal microbial composition. Higher species richness and diversity of the intestinal microbiota were observed in the alternate-feeding groups. A PCoA analysis showed that the samples clustered separately according to the feeding strategy, and among the three groups, the SO/FO group clustered relatively closer to the BT/FO group. The alternate feeding significantly decreased the abundance ofMycoplasmaand selectively enriched specific microorganisms, including short-chain fatty acid (SCFA)-producing bacteria, digestive bacteria (CorynebacteriumandSphingomonas), and several potential pathogens (DesulfovibrioandMycobacterium). Alternate feeding may maintain the intestinal microbiota balance by improving the connectivity of the ecological network and increasing the competitive interactions within the ecological network. The alternate feeding significantly upregulated the KEGG pathways of fatty acid and lipid metabolism, glycan biosynthesis, and amino acid metabolism in the intestinal microbiota. Meanwhile, the upregulation of the KEGG pathway of lipopolysaccharide biosynthesis indicates a potential risk for intestinal health. In conclusion, short-term alternate feeding between dietary lipid sources reshapes the intestinal microecology of the juvenile turbot, possibly resulting in both positive and negative effects.