*Purpose: A disrupted mucosal barrier in intestinal grafts can cause alloreactive complications such as rejection, inflammation and infection following intestinal transplantation (ITx). We hypothesize that RNAseq identifies novel genes and pathways associated with intestinal transplant complications in the postoperative course.

*Methods: To test our hypothesis, RNAseq experiments were performed on serial ITx biopsy samples from 5 healthy and 10 rejecting patients. Specifically, we studied the transcript abundance in rejecting allografts vs. healthy >6 months’ allografts, rejecting allografts at baseline vs. healthy allografts at baseline and “prerejection” allografts vs. healthy allografts. The group comparison analysis was performed using R-based Bioconductor software packages.

*Results: RNAseq revealed genome-wide transcriptomic differences between rejecting allografts vs. healthy >6 month’s allografts. Among 1535 differentially expressed genes (DEGs), 1026 were up- or 509-downregulated with statistical significance (False Discovery Rate (FDR)<0.01). We found that in the rejecting allografts, markedly higher expression genes related to inflammatory bowel disease, intestinal barrier viability, as well as genes associated with CD8+T cell-mediated cytotoxic responses were present. To understand the biological meaning of DEGs, Gene ontology (GO) and pathway analysis were conducted. The most prominent biological processes of GO were as follows: complement activation, regulation of immune response, leukocyte migration, and inflammatory responses. Similarly, we observed that genes related to inflammatory bowel disease and intestinal barrier viability were significantly upregulated in the rejecting allografts at baseline compared to healthy allografts at baseline. GO pathway analysis showed the DEGs for prerejection vs. healthy allografts were particularly enriched in inflammatory response, chemokine-mediated signaling pathway, and neutrophil degranulation.

*Conclusions: RNAseq of serial ITx biopsies identified dynamic transcriptomic differences over time. The expression of genes associated with inflammatory bowel disease and mucosal barrier homeostasis were markedly increased in intestinal allografts undergoing rejection, raising the possibility that RNAseq-based tissue immunomonitoring will also be capable of detecting acute rejection before it is clinically evident.

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