Paneth cell α-defensins mediate host defense and homeostasis at the intestinal mucosal surface. In mice, matrix metalloproteinase-7 (MMP7) converts inactive pro-α-defensins (proCrps) to bactericidal forms by proteolysis at specific proregion cleavage sites. MMP7(-/-) mice lack mature α-defensins in Paneth cells, accumulating unprocessed precursors for secretion. To test for activation of secreted pro-α-defensins by host and microbial proteinases in the absence of MMP7, we characterized colonic luminal α-defensins. Protein extracts of complete (organ plus luminal contents) ileum, cecum, and colon of MMP7-null and wild-type mice were analyzed by sequential gel permeation chromatography/acid-urea polyacrylamide gel analyses. Mature α-defensins were identified by N-terminal sequencing and mass spectrometry and characterized in bactericidal assays. Abundance of specific bacterial groups was measured by qPCR using group specific 16 S rDNA primers. Intact, native α-defensins, N-terminally truncated α-defensins, and α-defensin variants with novel N termini due to alternative processing were identified in MMP7(-/-) cecum and colon, and proteinases of host and microbial origin catalyzed proCrp4 activation in vitro. Although Paneth cell α-defensin deficiency is associated with ileal microbiota alterations, the cecal and colonic microbiota of MMP7(-/-) and wild-type mice were not significantly different. Thus, despite the absence of MMP7, mature α-defensins are abundant in MMP7(-/-) cecum and colon due to luminal proteolytic activation by alternative host and microbial proteinases. MMP7(-/-) mice only lack processed α-defensins in the small intestine, and the model is not appropriate for studying effects of α-defensin deficiency in cecal or colonic infection or disease.