This study was conducted to assess the effect of dietary supplementation of Muramidase 007 to broiler chickens on gastrointestinal functionality, evaluating growth performance, apparent ileal digestibility, intestinal histomorphology, vitamin A in plasma and cecal microbiota. A total of 480 one-day male chicks (Ross 308) were distributed in 16 pens allocated in 2 experimental diets: the control diet (CTR) without feed enzymes, coccidiostat or growth promoters, and the experimental diet (MUR): CTR supplemented with 35,000 units (LSU(F))/kg of the Muramidase 007. Digesta and tissue samples were obtained on days 9 and 36 of the study. A lower feed conversion ratio was observed in the MUR treatment. Apparent ileal digestibility of DM, organic matter and energy were improved by Muramidase 007. It was also observed that MUR improved digestibility of total fatty acids, mono-unsaturated fatty acids and poly-unsaturated fatty acids, and content of vitamin A in plasma at day 9 (P < 0.05). Histomorphological analysis of jejunum samples revealed no differences in the villus height or crypt depth; but a higher number of goblet cells and intraepithelial lymphocytes at day 36 with MUR. No differences were observed in plate counts of enterobacteria or Lactobacillus along the gastrointestinal tract, neither on the cecal short-chain fatty acids. An statistical trend was observed for reduction of cecal clostridia at day 9 for MUR. Analysis of cecal microbiota structure by 16S rRNA gene sequencing revealed relevant changes correlated to age. At day 9, broilers receiving MUR showed decreased alpha diversity compared to CTR that was not detected at day 36. Changes in specific taxonomic groups with an increase in Lactobacillus genus were identified. In conclusion, evaluation of the variables in this study indicates that dietary Muramidase 007 contributes to improve feed conversation ratio and gastrointestinal function in broiler chickens. Effects could have been mediated by slight shifts observed in the intestinal microbiota. More studies are guaranteed to fully understand the mechanisms involved.