BACKGROUND AND AIMS: Fistula formation is a common and debilitating complication in Crohn's disease (CD). CD-associated fistula carcinomas (Fi-Cas), though rare, pose diagnostic and prognostic challenges. This study aims to identify disease-defining immune cell subsets in CD-associated Fi-Cas. METHODS: The study included tissue samples from 10 CD patients with Fi-Cas, 7 with CD-associated fistulas, and 6 with sporadic colorectal cancer (CRC). The main tumor (MT), infiltration front, and non-involved areas were analyzed in tumor samples. A 36-marker panel was employed to define the immune landscape using imaging mass cytometry. Samples were processed, stained, and analyzed for immune cell compositions, cell-cell interactions, and spatial microenvironments. RESULTS: The immune infiltrate in Fi-Cas shared similarities with both CD fistulas and CRC. Fi-Ca samples exhibited high levels of neutrophils, B cells, and CD163high macrophages. CRC MT samples showed an increased presence of intraepithelial CD8+ lymphocytes and CD163low macrophages. Cleaved Caspase-3 levels were highest in CRC MT samples, correlating positively with CD163low macrophages and cytotoxic T cells. In contrast, Fi-Ca MT samples showed a negative correlation between cleaved Caspase-3 and cytotoxic T cells. Analysis of cellular microenvironments and dimensionality reduction clustering based on immune cell frequencies indicated Fi-Cas to exhibit a mixture of immune cell characteristics from both CD fistulas and CRC. CONCLUSIONS: The immune landscape of CD-associated Fi-Cas exhibits features of both CD fistulas and CRC, suggesting a complex pathogenesis influenced by chronic inflammation. Our data suggest that Fi-Cas represent a unique cancer subtype, that requires further analysis to develop targeted therapeutic strategies.