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This study was conducted in two stages to investigate the potential of multi-enzyme supplementation on the nutrient digestibility, growth performance, and gut microbial composition of pigs. In stage 1, effects of multi-enzyme complex (xylanase, α-amylase, β-glucanase, and protease) supplementation on the ileal and total tract dry matter (DM) digestibility of feed-stuffs were investigated with in vitro two-stage and three-stage enzyme incubation methods. A wide range of feed ingredients, namely, corn meal, wheat meal, soybean meal, fish meal, Oriental herbal extract, Italian rye-grass (IRG) and peanut hull were used as substrates. Supplementation of the multi-enzyme complex increased (P < 0.05) the digestibility of the Oriental herbal extract and corn meal. In stage 2, in vivo animal studies were performed to further investigate the effects of the dietary multi-enzyme complex on the nutrient utilization, growth performance, and fecal microbial composition of pigs. A total of 36 weaned pigs were fed corn- and soybean meal-based diets without (control) and with the multi-enzyme complex (treatment) for 6 weeks. Fecal samples were collected from 12 pigs to analyze the microbial communities by using DNA sequencing and bioinformatics tools. Multi-enzyme supplementation had no effect on apparent digestibility of nutrients and growth performance of pigs compared to control. Taxonomic analysis of the fecal samples indicated that the bacteria in both control and treatment samples predominantly belonged to Firmicutes and Bacteroidetes. In addition, the proportion of the phylum Firmicutes was slightly higher in the treatment group. At the genus level, the abundance of Treponema and Barnesiella increased in the treatment group; whereas the numbers ofthe genera including Prevotella, Butyricicoccus, Ruminococcus and Succinivibrio decreased in the treatment group. These results suggest that multi-enzyme supplementation with basal diets have the potential to improve nutrient digestibility and modify microbial communities in the hind-gut of pigs. [ABSTRACT FROM AUTHOR]