Enterotoxigenic Bacteroides fragilis (ETBF) produces an ≈ 20 kDa B. fragilis enterotoxin (BFT), which plays an essential role in mucosal inflammation. Lipocalin (Lcn)-2, a siderophore-binding antimicrobial protein, is critical for control of bacterial infection; however, expression of Lcn-2 in BFT-exposed intestinal epithelial cells has not been elucidated. In the present study, stimulation of human intestinal epithelial cells with BFT resulted in the upregulation of Lcn-2 expression that was a relatively late response of intestinal epithelial cells compared with human β-defensin (hBD)-2 expression. The upregulation of Lcn-2 was dependent on AP-1 but not on NF-κB signaling. Lcn-2 induction via AP-1 was regulated by mitogen-activated protein kinases (MAPKs) including ERK and p38. Lcn-2 was secreted from the apical and basolateral surfaces in BFT-treated cells. These results suggest that a signaling pathway involving MAPKs and AP-1 is required for Lcn-2 induction in intestinal epithelial cells exposed to BFT, after which the secreted Lcn-2 may facilitate antimicrobial activity within ETBF-infected mucosa.