Objective To investigate the mechanism of action of moxibustion in improving visceral hypersensitivity in rats with diarrhea-predominant irritable bowel syndrome (IBS-D) by observing the intervention effect of moxibustion on the microRNA-24 (miR-24)/serotonin transporter (SERT)/5-hydroxytryptamine (5-HT) pathway.Methods Sprague-Dawley rats were randomly divided into normal group, model group, and moxibustion group, with 12 rats in each group. A rat model of IBS-D was established by mother-child separation combined with acetic acid stimulation and chronic restraint stress. The rats in the moxibustion group were given moxibustion at "Tianshu" and "Shangjuxu" at both sides for 20 minutes, once a day for 7 consecutive days. Body weight, loose stool rate, and minimum volume threshold of abdominal withdrawal reflex (AWR) were measured on days 34, 45, and 53; HE staining was used to observe the histopathological changes of the colon; ELISA was used to measure the changes in the levels of 5-HT, SERT, serotonin receptor 4 (5-HT4R), calcitonin gene-related peptide (CGRP), and substance P (SP) in serum, midbrain, and colon tissue; Western blot and RT-PCR were used to measure the protein and mRNA expression levels of SERT, 5-HT4R, CGRP, and SP in the midbrain and colon tissue, and RT-PCR was used to measure the expression level of miR-24 in the midbrain and colon tissue. Results Compared with the normal group, the model group had a significant increase in loose stool rate (P<0.05), a significant reduction in the minimum volume threshold of AWR (P<0.05), significant increases in the levels of 5-HT, 5-HT4R, CGRP, and SP (P<0.05) and a significant reduction in the level of SERT in serum, midbrain, and colon tissue (P<0.05), a significant increase in the expression level of miR-24 (P<0.05), significant reductions in the protein and mRNA expression levels of SERT (P<0.05), and significant increases in the protein and mRNA expression levels of 5-HT4R, CGRP, and SP (P<0.05). Compared with the model group, the moxibustion group had a significant reduction in loose stool rate (P<0.05), a significant increase in the minimum volume threshold of AWR (P<0.05), significant reductions in the levels of 5-HT, 5-HT4R, CGRP, and SP (P<0.05) and a significant increase in the level of SERT in serum, midbrain, and colon tissue (P<0.05), a significant reduction in the expression level of miR-24 (P<0.05), significant increases in the protein and mRNA expression levels of SERT (P<0.05), and significant reductions in the protein and mRNA expression levels of 5-HT4R, CGRP, and SP (P<0.05). Conclusion Moxibustion improves visceral hypersensitivity in IBS-D rats possibly by downregulating the expression of miR-24, promoting the reuptake of 5-HT by SERT, and reducing the level of 5-HT.