Objective To investigate the effect and possible mechanism of acupuncture on small intestine propulsion rate and levels of 5-hydroxytryptamine (5-HT), vasoactive intestinal peptide (VIP), and substance P (SP) in serum and the colon in a rat model of constipation-predominant irritable bowel syndrome (IBS-C). Methods Among 60 clean healthy Sprague-Dawley rats, 10 were randomly selected as normal control group and 50 rats were used to establish a model of IBS-C by the comprehensive cold-restraint-starvation method. After model evaluation, 30 rats were selected and divided into model group, four-single-acupoint group, and non-meridian/acupoint group using a random number table, with 10 rats in each group. The rats were given activated carbon suspension by gavage to observe small intestine propulsion rate; ELISA was used to measure the serum levels of 5-HT, VIP, and SP, and immunohistochemistry was used to measure their content in the colon. Results Compared with the normal control group, the model group had a significant reduction in small intestine propulsion rate (P<0.05), and compared with the model group, the four-single-acupoint group had a significant increase in small intestine propulsion rate (P<0.05). Compared with the normal control group, the model group had significant increases in 5-HT and VIP and a significant reduction in SP in serum and the colon (P<0.05); compared with the model group and the non-meridian/acupoint group, the four-single-acupoint group had significant reductions in 5-HT and VIP and a significant increase in SP in serum and the colon (P<0.05). Conclusion Acupuncture can significantly increase the small intestine propulsion rate of IBS-C rats, possibly by downregulating 5-HT and VIP and upregulating SP in serum and the colon.