Unexpected environmental and social stimuli could trigger stress. Although coping with stress is essential for survival, long-term stress impacts visceral functions, and therefore, it plays a role in the development and exacerbation of symptoms of gastrointestinal/urogenital disorders. The aim of this study is to characterize the role of corticosterone in stress-sensitized colon-bladder cross-talk, a phenomenon presumed to underlie the comorbidity of functional bowel and bladder disorders. Cystometry and protein/mRNA expression in the lumbosacral dorsal horn (L6-S1) in response to intracolonic mustard oil (MO) instillation were analyzed in female Wistar-Kyoto rats subjected to water avoidance stress (WAS; 1 h/day for 10 days) or sham stress (WAsham). Whereas it had no effect on baseline-voiding function, chronic stress upregulated plasma corticosterone concentration and dorsal horn spinal p90 ribosomal S6 kinase 2 (RSK2) protein/mRNA levels, and RSK2 immunoreactivity colocalized with NeuN-positive neurons. Intracolonic MO dose-dependently decreased intrercontraction intervals and threshold pressure, provoked spinal RSK2 and NR2B phosphorylation, and enhanced PSD-95-RSK2 and PSD-95-NR2B coupling. Intrathecal kaempferol (a RSK2 activation antagonist; 30 min before MO instillation), bilateral adrenalectomy (7 days prior the stress paradigm), and subcutaneous RU-38486 (a glucocorticoid receptor antagonist; 30 min daily before stress sessions), but not RU-28318 (a mineralocorticoid receptor antagonist), attenuated MO-induced bladder hyperactivity, protein phosphorylation, and protein-protein interactions in the WAS group. Our results suggest that stress-associated glucocorticoid release mediates WAS-dependent sensitization of colon-bladder cross-talk via the spinal RSK2/PSD-95/NR2B cascade and offer a possibility for developing pharmacological strategies for the treatment of stress-related pelvic pain.