AbstractThis research was conducted to assess the effects of replacing fish meal (FM) with distillers’ dried grains with solubles (DDGS) at various levels (D1 [0% of total diet] as control diet, D2 [2%], D3 [4%], D4 [6%], D5 [8%], D6 [12%], and D7 [16%]) on the growth performance and gut microbiota in juvenile Pacific whiteleg shrimp Litopenaeus vannamei. Shrimp with an initial weight of 0.23 ± 0.00 g (mean ± SD) were stocked (40 shrimp/tank) in seven different tanks, with three replicates of each treatment group. Shrimp were fed their respective experimental diets for 56 d. Afterwards, an Illumina‐based sequencing method was used to examine the intestinal bacterial composition of the shrimp. The results showed an enhancement of growth performance, including final weight and weight gain, in shrimp that were fed DDGS diets. In addition, the feed conversion ratio was significantly lower in shrimp that were fed D6 and D8 than in shrimp that received D0. Survival rates ranged from 76.6% to 92.50%. The richness estimates (Chao 1 and the abundance‐based coverage estimator) and diversity indices (Shannon and Simpson) of the microbial community were affected when DDGS were included in the shrimp diet. Beneficial bacteria, including Proteobacteria, Bacteroidetes, and Actinobacteria, dominated the gut microbiota at the phylum level for shrimp that were fed DDGS diets. The number of operational taxonomic units obtained ranged from 783 to 920. These results suggest that replacing FM with DDGS at 8% (D5) in the total shrimp diet could enhance the growth performance and affect gut microbiota composition in Pacific whiteleg shrimp.