Lipocalin 2 (Lcn2), also called neutrophil gelatinase B-associated lipocalin (NGAL), is an anti-microbial peptide originally identified in neutrophil granules. Although Lcn2/NGAL expression is increased in the inflamed intestinal tissues of patients with inflammatory bowel disease, the role of Lcn2/NGAL in the development of intestinal inflammation remains unclear. Here we investigated the role of Lcn2/NGAL in intestinal inflammation using a spontaneous mouse colitis model, interleukin-10 knock out (IL-10 KO) mice. Lcn2 expression in the colonic tissues of IL-10 KO mice increased with the development of colitis. Lcn2/IL-10 double-KO mice showed a more rapid onset and development of colitis compared to IL-10 KO mice. Lcn2 enhanced phagocytic bacterial clearance in macrophages in vitro after infection with Escherichia coli. Transfer of Lcn2-repleted macrophages prevented the development of colitis in Lcn2/IL-10 double-KO mice in vivo. Our findings revealed that Lcn2 prevents the development of intestinal inflammation. One crucial factor seems to be the enhancement of phagocytic bacterial clearance in macrophages by Lcn2.