Inflammatory bowel disease (IBD) is commonly treated via enteral administration, which suffers from limited drug stability, low bioavailability, and short retention time. Parenteral administration—particularly intraperitoneal injection—offers a promising alternative by bypassing the gastrointestinal tract and enhancing drug delivery to the intestines. However, intraperitoneally administered drugs primarily localize on the serosal rather than the mucosal side, where inflammation occurs. It remains unclear whether drug nanoparticles can penetrate the intestinal wall or modulate inflammation indirectly via lymphoid organs. Here, we employed tacrolimus (FK506)-loaded core–shell nanoparticles (FK506-Core/Shell) to investigate the feasibility and mode of action of parenteral administration for IBD treatment, with comparison to uncoated FK506-Core nanoparticles. Among three parenteral routes—intravenous, subcutaneous, and intraperitoneal injection—we found that intraperitoneal administration led to stronger accumulation of nanoparticles, especially FK506-Core/Shell, in the colon, with detectable signals also observed in mesenteric lymph nodes (mLNs) and the spleen. Following intraperitoneal injection, these nanoparticles were effectively internalized by antigen-presenting cells in the peritoneal fluid, accompanied by changes in cell populations, suggesting cell-mediated trafficking in vivo. Furthermore, nanoparticle signals were detected on the mucosal side of the intestine, as well as within mLNs and the spleen, supporting localized and systemic anti-inflammatory effects. Treatment with FK506-Core/Shell significantly ameliorated dextran sulfate sodium (DSS)-induced colitis in mice, correlating with suppressed immune activation and induction of tolerance. These findings support the potential of intraperitoneally delivered nanotherapeutics as an effective strategy for IBD treatment.