The aim of this study was to investigate the effect of grain type on performance and gut microbiota composition in broiler chickens under a necrotic enteritis (NE) challenge. Broilers were fed 3 different diets based on wheat, barley or rye, and were orally administered Eimeria and Clostridium perfringens to induce subclinical NE. The NE challenge significantly decreased weight gain, feed intake and counts of Bacillus and Ruminococcus bacteria in the caecal contents. Birds fed the rye based diets showed significantly reduced weight gain and feed conversion compared to the other diets. Rye fed birds also had the highest fluorescein isothiocyanate-dextran (FITC-d) marker in their serum and the lowest concentration of Ruminococcus bacteria and acetic acid in caecal contents.