BACKGROUND: Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by persistent inflammation of the colon. The specific cause of UC is still not fully understood, but this condition is believed to arise from a combination of environmental, genetic, microbial, and immune factors. This study aimed to explore the specific roles of macrophages and fibroblasts in UC pathogenesis, focusing on their interactions and contributions to disease progression. METHODS: We utilized single-cell RNA sequencing (scRNA-seq) to analyze macrophages and fibroblasts in peripheral blood and colon biopsy samples from UC patients. Bulk RNA sequencing and spatial transcriptomic data from the Gene Expression Omnibus (GEO) database and flow cytometry and multiplex immunohistochemistry (mIHC) data were used for validation. Statistical analyses were performed to assess the correlation between cell abundance and disease severity. RESULTS: Macrophages and fibroblasts were identified as key communication hubs in UC; specifically, SPP1 + macrophages and CHI3L1 + fibroblasts were significantly enriched at the sites of inflammation. These cells are strongly correlated with disease severity and orchestrate inflammatory responses within the intestinal immune microenvironment, contributing to UC-associated colorectal cancer. CONCLUSIONS: Our study identified SPP1 + macrophages and CHI3L1 + fibroblasts as key contributors to UC pathogenesis. These cells are enriched in inflammatory sites, are correlated with disease severity, and play a role in UC-associated colorectal cancer, providing new insights into UC mechanisms.