Crohn's disease (CD)-associated adherent invasive Escherichia coli (AIEC) has been implicated as a causative or contributory factor in CD pathology. MicroRNA let-7b/Toll-like receptor 4 (TLR-4) signaling pathways may play an important role in microbiota-mucosa interactions. We aimed to investigate whether AIEC influences IECs' function of expressing TLR4, and the potential role of let-7b in regulating AIEC induced gut inflammation. Wild type (WT) and interleukin-10 knockout (IL-10 KO) mice in specific pathogen free (SPF) housing were infected by AIEC LF82, and IL-10 KO mice were treated with pre-let7b or anti-let-7b. Besides, T84 cells were infected by AIEC LF82 or E. coli K-12, and treated with let-7b mimics or inhibitor. let-7b/TLR4 signaling pathway was investigated and the therapeutic effects of let-7b treatment on colitis in IL-10-mice and on cytokines release in T84 were assessed. We found AIEC elicited/exacerbated colitis in WT/IL-10 KO mice, and promoted proinflammatory cytokines release in T84 cells that correlated with increased TLR4 expression and decreased let-7b. Overexpression of let-7b significantly ameliorated the severity of colitis in AIEC infected IL-10 KO mice, and reduced secretion of cytokines in AIEC-infected T84 cells via regulating TLR4 expression. Besides, overexpression of TLR4 caused by inhibition of let-7b led to increased proinflammatory cytokines release in K-12 infected T84 cells. These results suggest AIEC instigates excessive mucosal immune response against gut microbiota via let-7b/TLR4 signaling pathway. Let-7b may be a potential therapeutic target of CD, especially for CD with AIEC infection.