κ-Casein glycomacropeptide (GMP) exerts anti-inflammatory and immune modulatory effects. A bovine GMP concentrate and its in vitro digestion product were obtained. GMP was also microencapsulated with phycocolloids and further digested. These products were tested in three-dimensional (3D) and open monolayer two-dimensional (2D) mouse jejunal organoids. Almost no effect was observed on the 2D organoids. In 3D organoids, GMP induced intestinal proliferation (Axin2, Pcna) and differentiation (Vil1, Alpl) genes together with Muc3, antibacterial genes (Lyz1, Pla2g2a), and Cxcl1. GMP also induced interferon I defense genes (Ifnb1, Ifr3, Oas2, Oas3, Rnasel) under basal conditions and in TNF-stimulated organoids. In vitro digestion abrogated the effects of GMP and induced new genes (Lgr5, Olfm4, and Lct). In TNF-stimulated organoids, digested GMP repressed multiple genes. Encapsulation largely preserved the GMP effects. In conclusion, GMP showed differential effects in 3D and 2D organoids. The effects of digestion peptides were also different, suggesting distinct potential as functional foods.