Intercalated cells of the collecting duct (CD) are critical for acid-base homeostasis and innate immune defense of the kidney. Little is known about the impact of acidosis on innate immune defense in the distal nephron. Urinary tract infections are mainly due to Escherichia coli and are an important risk factor for development of chronic kidney disease. While the effect of urinary pH on growth of E. coli is well established, in this study we demonstrate that acidosis increases urine antimicrobial activity due, at least in part, to induction of cathelicidin expression within the CD. Acidosis was induced in rabbits by adding NH₄Cl to the drinking water and reducing food intake over 3 days or by casein supplementation. Microdissected CDs were examined for cathelicidin mRNA expression and antimicrobial activity and cathelicidin protein levels in rabbit urine were measured. Cathelicidin expression in CD cells was detected in kidney sections. CDs from acidotic rabbits expressed three times more cathelicidin mRNA than those isolated from normal rabbits. Urine from acidotic rabbits had significantly more antimicrobial activity (versus E. coli) than normal urine and most of this increased activity was blocked by cathelicidin antibody. The antibody had little effect on antimicrobial activity of normal urine. Urine from acidotic rabbits had at least twice the amount of cathelicidin protein as did normal urine. We conclude that metabolic acidosis not only stimulates CD acid secretion but also induces expression of cathelicidin and thereby enhancing innate immune defense against urinary tract infections via induction of antimicrobial peptide expression.