Background: Irritable Bowel Syndrome (IBS), as a functional gastrointestinal disorder, is characterized by abdominal pain and distension. Recent studies have shown that acupuncture treatment improves symptoms of diarrhea-predominant irritable bowel syndrome (IBS-D) by altering networks in certain brain regions. However, few studies have used resting-state functional magnetic resonance imaging (fMRI) to compare altered resting-state inter-network functional connectivity in IBS-D patients before and after acupuncture treatment. Objective: To analyze altered resting-state inter-network functional connectivity in IBS-D patients before and after acupuncture treatment. Methods: A total of 74 patients with IBS-D and 31 healthy controls (HCs) were recruited for this study. fMRI examination was performed in patients with IBS-D before and after acupuncture treatment, but only at baseline in HCs. Data on the left frontoparietal network (LFPN), default mode network (DMN), salience network (SN), ventral attention network (VAN), auditory network (AN), visual network (VN), sensorimotor network (SMN), dorsal attention network (DAN), and right frontoparietal network (RFPN) were subjected to independent component analysis (ICA). The functional connectivity values of inter-network were explored. Results: Acupuncture decreased irritable bowel syndrome symptom severity score (IBS-SSS) and Hamilton Anxiety Scale (HAMA). It also ameliorated symptoms related to IBS-D. Notably, functional connectivity between AN and VAN, SMN and DMN, RFPN and VAN in IBS-D patients after acupuncture treatment was different from that in HCs. Furthermore, there were differences in functional connectivity between DMN and DAN, DAN and LFPN, DMN and VAN before and after acupuncture treatment. The inter-network changes in DMN-VAN were positively correlated with changes in HAMA, life influence degree, and IBS-SSS in IBS-D. Conclusion: Altered inter-network functional connectivity is involved in several important hubs in large-scale networks. These networks are altered by acupuncture stimulation in patients with IBS-D.