Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder characterized by abdominal pain and alterations in bowel habits. Current treatments for IBS are unsatisfactory due to its multifactorial pathogenesis involving the microbiota-gut-brain axis. Lactobacillus plantarum PS128 (PS128) was reported to exhibit neuromodulatory activity which may be beneficial for improving IBS. This study aimed to investigate the effect of PS128 on visceral hypersensitivity (VH) and the gut-brain axis using a 5-hydroxytryptophan (5-HTP)-induced VH rat model without colonic inflammation induction, mimicking the characteristics of IBS. Male Sprague-Dawley rats were administered with PS128 (10 CFU in 0.2 mL saline/rat/day) or saline (0.2 mL saline/rat/day) for 14 days. Colorectal distension (CRD) with simultaneous electromyography recording was performed 30 min before and 30 min after the 5-HTP injection. Levels of neuropeptides and neurotrophins were analyzed. PS128 significantly reduced VH induced by the 5-HTP injection and CRD. Neurotransmitter protein levels, substance P, CGRP, BDNF, and NGF, were decreased in the dorsal root ganglion but increased in the spinal cord in response to the 5-HTP injection; PS128 reversed these changes. The hypothalamic-pituitary-adrenal axis was modulated by PS128 with decreased corticosterone concentration in serum and the expression of mineralocorticoid receptors in the amygdala. Oral administration of PS128 inhibited 5-HTP-induced VH during CRD. The ameliorative effect on VH suggests the potential application of PS128 for IBS.