Carbonic anhydrase 2 regulates acid-base homeostasis, and recent findings have indicated a correlation between cellular control of acid-base status and the innate defense of the kidney. Mice deficient in carbonic anhydrase 2 (Car2^−/− mice) have metabolic acidosis, impaired urine acidification, and are deficient in normal intercalated cells. The objective of the present study was to evaluate the biological consequences of carbonic anhydrase 2 deficiency in a murine model of pyelonephritis. Infection susceptibility and transcription of bacterial response components in Car2^−/− mice were compared with wild-type littermate controls. Car2^−/− mice had increased kidney bacterial burdens along with decreased renal bacterial clearance after inoculation compared with wild-type mice. Standardization of the urine pH and serum HCO₃⁻ levels did not substantially alter kidney infection susceptibility between wild-type and Car2^−/− mice; thus, factors other than acid-base status are responsible. Car2^−/− mice had significantly increased neutrophil-gelatinase-associated lipocalin mRNA and protein and expression at baseline and a marked decreased ability to upregulate key bacterial response genes during pyelonephritis. Our findings provide in vivo evidence that supports a role for carbonic anhydrase 2 and intercalated cells in promoting renal bacterial clearance. Decreased carbonic anhydrase expression results in increased antimicrobial peptide production by cells other than renal intercalated cells, which is not sufficient to prevent infection after a bacterial challenge.