Human β-defensin 1 (hBD-1) is an antimicrobial peptide expressed by epithelia and hematopoietic cells. We demonstrated recently that hBD-1 shows activity against enteric commensals and Candida species only after its disulfide bonds have been reduced by thioredoxin (TRX) or a reducing environment. Here we show that besides TRX, glutaredoxin (GRX) is also able to reduce hBD-1, although with far less efficacy. Moreover, living intestinal and lymphoid cells can effectively catalyze reduction of extracellular hBD-1. By chemical inhibition of the TRX system or specific knockdown of TRX, we demonstrate that cell-mediated reduction is largely dependent on TRX. Quantitative PCR in intestinal tissues of healthy controls and inflammatory bowel disease patients revealed altered expression of some, although not all, redox enzymes, especially in ulcerative colitis. Reduced hBD-1 and TRX localize to extracellular colonic mucus, suggesting that secreted or membrane-bound TRX converts hBD-1 to a potent antimicrobial peptide in vivo.