Barley, increasingly recognized for its health benefits, contains bioactive compounds like beta-glucans and (poly)phenols. Newly developed purple barley varieties, enriched with anthocyanins, offer potential gut health benefits. This study examined the effects of a hull-less, purple-grain barley genotype, consumed as whole-grain or isolated fractions (bran and endosperm), on gut microbiota and inflammation in a murine model. Fifty male and female BALB/cB&J mice were assigned to five diets over six weeks: standard diet (SD), rice diet (RD), whole-grain barley (WGB), anthocyanin-rich barley bran (BB), and beta-glucan-rich endosperm (PG). The BB diet triggered anti-inflammatory signals as it reduced IFN-γ and IL-4 in females, lowered TNF-α in both sexes, and decreased C-Reactive Protein (CRP) in males compared to SD. The PG diet improved gut barrier integrity by lowering LPS-binding protein levels. Barley-based diets enhanced gut microbiota diversity, particularly, by increasing beneficial bacteria like