Single-cell spatial transcriptomics offers transformative potential for understanding inflammatory bowel disease, yet dissociation-based approaches lose spatial context and suffer from cellular dropout. In ulcerative colitis (UC), nearly half of patients fail vedolizumab (VDZ), and the cellular underpinnings of resistance remain poorly defined. Imaging-based single-cell spatial transcriptomics (iSCST) on formalin-fixed, paraffin-embedded (FFPE) tissue can overcome these barriers by profiling archived biopsies at subcellular resolution while preserving tissue architecture.We deployed an optimized iSCST framework on FFPE colon biopsies from UC, immune checkpoint inhibitor colitis, and healthy controls, including specimens archived up to eleven years. Three commercial platforms were benchmarked, and a custom Xenium gene panel was designed for IBD mucosa. Spatial cellular neighborhoods were defined using CellCharter, and pre-treatment VDZ signatures were validated in an internal replication cohort and an external bulk transcriptomic dataset. The optimized panel resolved transcriptionally distinct fibroblast, epithelial, and immune subsets, identifying colitis-specific neighborhoods composed of inflammation-associated fibroblasts (IAFs), monocytes, and neutrophils. Critically, VDZ resistance was encoded not by individual cell types but by spatial organization into discrete neighborhoods. Responders were enriched for an epithelial neighborhood, while non-responders segregated into two mechanistically distinct archetypes: an innate archetype defined by an IAF–monocyte–neutrophil neighborhood, and an adaptive archetype defined by a gut-associated lymphoid tissue (GALT) neighborhood characterized by activated dendritic cells within organized lymphoid aggregates. These activated dendritic cells, spatially colocalized within GALT structures, suggest a locally orchestrated adaptive immune program sustaining mucosal inflammation independently of α4β7-integrin–mediated lymphocyte trafficking — providing a compelling mechanistic basis for VDZ resistance in this subset. Both archetypes were validated across cohorts and platforms, and baseline disease severity did not differ between groups. This framework redefines VDZ resistance as the product of two distinct cellular ecosystems within archived FFPE tissue, opening a path toward spatial biomarker-guided therapy selection.