Background Many patients with intestinal failure require intestinal transplantation (ITx) to survive. Acute cellular rejection poses a challenge in ITx because its biologic components are incompletely understood. New methodologies for its integrative and longitudinal analysis are needed. Methods In this study, we characterized episodes of acute cellular rejection in ITx recipients using a noninvasive proteomic analysis. Ostomy effluent was obtained from all patients undergoing ITx at University of California, Los Angeles from July 2008 to September 2009 during surveillance endoscopies in the first 8 weeks post-ITx. Effluent was analyzed using 17-plex Luminex technology and matrix-assisted laser desorption/ionization proteomics. Results Of 56 ostomy effluent samples from 17 ITx recipients, 14% developed biopsy-proven rejection at a median of 25 days post-ITx. Six had mild rejection, two were indeterminate for rejection, and no graft loss was seen in the first 3 months posttransplantation. Effluent levels of five innate immune cytokines were elevated in the posttransplantation phase: granulocyte colony-stimulating factor, interleukin-8, tissue necrosis factor-α, interleukin-1β, and interferon-γ. Proteomic analysis revealed 17 protein features differentially seen in rejection, two identified as human neutrophil peptide 1 and 2. This was confirmed by the presence of human neutrophil peptide-positive lamina propria neutrophils in biopsy tissue samples. Conclusions Proteomic and cytokine analysis of ostomy effluents suggests an early and unappreciated role of innate immune activation during rejection.