Psychological stress has been previously reported to worsen symptoms of inflammatory bowel disease (IBD). Similarly, intestinal tertiary lymphoid organs (TLOs) are associated with more severe inflammation. While there is active debate about the role of TLOs and stress in IBD pathogenesis, there are no studies investigating TLO formation in the context of psychological stress. Our mouse model of Crohn's disease-like ileitis, the SAMP1/YitFc (SAMP) mouse, was subjected to 56 consecutive days of restraint stress (RS). Stressed mice had significantly increased colonic TLO formation. However, stress did not significantly increase small or large intestinal inflammation in the SAMP mice. Additionally, 16S analysis of the stressed SAMP microbiome revealed no genus-level changes. Fecal microbiome transplantation into germ-free SAMP mice using stool from unstressed and stressed mice replicated the behavioral phenotype seen in donor mice. However, there was no difference in TLO formation between recipient mice. Stress increased the TLO formation cytokines interleukin-23 (IL-23) and IL-22 followed by up-regulation of antimicrobial peptides. SAMP × IL-23r-/- (knockout [KO]) mice subjected to chronic RS did not have increased TLO formation. Furthermore, IL-23, but not IL-22, production was increased in KO mice, and administration of recombinant IL-22 rescued TLO formation. Following secondary colonic insult with dextran sodium sulfate, stressed mice had reduced colitis on both histology and colonoscopy. Our findings demonstrate that psychological stress induces colonic TLOs through intrinsic alterations in IL-23 signaling, not through extrinsic influence from the microbiome. Furthermore, chronic stress is protective against secondary insult from colitis, suggesting that TLOs may function to improve the mucosal barrier. Keywords: IL-23; Tertiary Lymphoid Organs; inflammatory bowel disease; microbiome; stress.