Gram-negative pathogenic bacteria activate mucosal inflammation through lipopolysaccharide (LPS) binding to intestinal toll-like receptor 4 (TLR4). This study tested whether human milk oligosaccharides (HMOSs) influence pathogenic Escherichia coli-induced interleukin (IL)-8 release by intestinal epithelial cells (IECs), identified specific proinflammatory signaling molecules modulated by HMOSs, and specified the active HMOS and determined its mechanism of action. Inflammation models were IECs invaded by type 1 pili enterotoxigenic E. coli (ETEC) in vitro, with T84 modeling mature, and H4 modeling immature, IECs. HMOSs attenuated LPS-dependent induction of IL-8 caused by ETEC, UPEC, and AIEC infection, and suppressed CD14 transcription and translation. CD14 knockdown recapitulated HMOS-induced attenuation. Overexpression of CD14 increased the inflammatory response to ETEC and sensitivity to inhibition by HMOSs. 2’-fucosyllactose (2’-FL) activity was equivalent to total HMOSs in vitro and in vivo. HMOSs and 2’-FL directly inhibit LPS mediated inflammation during ETEC invasion of T84 and H4 IECs through attenuation of CD14 induction. CD14 expression mediates LPS-TLR4 stimulation of the ‘macrophage migration inhibitory factors’ inflammatory pathway via SOCS 2/STAT 3/NF-κB. HMOS and 2’-FL inhibition of inflammation supports their functioning as components of an innate immune system whereby the mother’s milk quenches hyper-inflammation of the neonatal gut, and suggests their clinical utility.