Irritable bowel syndrome with diarrhea (IBS-D) is a chronic and relapsing inflammatory disorder in which pathogenesis has been shown to be in part the result of miRNA-mediated signaling. Here, we investigated the alleviatory role of miR-16 in IBS-D. First, we established an IBS-D mouse model using colonic instillation of acetic acid and developed an IBS-D cell model using lipopolysaccharide exposure. The experimental data demonstrated that miR-16 was underexpressed in the serum of IBS-D patients, as well as in the colorectal tissues of IBS-D mouse models and lipopolysaccharide-exposed intestinal epithelial cells. Next, miR-16 and TLR4 were overexpressed or inhibited to characterize their roles in the viability and apoptosis of intestinal epithelial cells, inflammation, and epithelial tight junction. We found that miR-16 overexpression increased the viability of intestinal epithelial cells, maintained tight junction integrity, and inhibited cell apoptosis and inflammation. We showed that miR-16 targeted TLR4 and inhibited the TLR4/NF-κB signaling pathway. Additionally, inhibition of NF-κB suppressed the long noncoding RNA XIST, thereby promoting enterocyte viability, inhibiting apoptosis and cytokine production, and maintaining tight junction integrity. In vivo experiments further verified the alleviatory effect of miR-16 on IBS-D symptoms in mice. Taken together, we conclude that miR-16 downregulates XIST through the TLR4/NF-κB pathway, thereby relieving IBS-D. This study suggests that miR-16 may represent a potential target for therapeutic intervention against IBS-D.