Clostridium difficile infection (CDI) is principally responsible for hospital acquired, antibiotic-induced diarrhea and colitis and represents a significant financial burden on our healthcare system. Little is known about C. difficile proliferation requirements, and a better understanding of these parameters is critical for development of new therapeutic targets. In cell lines, C. difficile toxin B has been shown to inhibit Na(+)/H(+) exchanger 3 (NHE3) and loss of NHE3 in mice results in an altered intestinal environment coupled with a transformed gut microbiota composition. However, this has yet to be established in vivo in humans. We hypothesize that C. difficile toxin inhibits NHE3, resulting in alteration of the intestinal environment and gut microbiota. Our results demonstrate that CDI patient biopsy specimens have decreased NHE3 expression and CDI stool has elevated Na(+) and is more alkaline compared with stool from healthy individuals. CDI stool microbiota have increased Bacteroidetes and Proteobacteria and decreased Firmicutes phyla compared with healthy subjects. In vitro, C. difficile grows optimally in the presence of elevated Na(+) and alkaline pH, conditions that correlate to changes observed in CDI patients. To confirm that inhibition of NHE3 was specific to C. difficile, human intestinal organoids (HIOs) were injected with C. difficile or healthy and CDI stool supernatant. Injection of C. difficile and CDI stool decreased NHE3 mRNA and protein expression compared with healthy stool and control HIOs. Together these data demonstrate that C. difficile inhibits NHE3 in vivo, which creates an altered environment favored by C. difficile.