Adhesive arachnoiditis (AA) is a rare form of chronic degenerative pathology associated with persistent inflammation in the arachnoid matter of the spinal cord. Despite the existing knowledge, the detailed pathological mechanisms underlying AA are not fully understood. This study aimed to elucidate through comprehensive single nuclei RNA sequencing (snRNA-seq) to delineate the transcriptomic landscape of AA. From six arachnoid membrane samples, a total of 52,886 cells met the quality control standards for analysis. The main cell populations identified with specific gene markers were as follows: fibroblasts, glial cells, microglial cells, endothelial cells, mural cells, plasma cells, and T cells. Downstream analysis of fibroblasts, glial cells, and microglial cells was performed. Notably, fibroblast subsets 1 and 3 demonstrated a strong association with AA. Among them, subcluster 3 demonstrated elevated expression of genes COL1A1, COL3A1, and FN1, indicative of enhanced Wnt/β-catenin and extracellular matrix (ECM) synthesis pathways. Subcluster 3 was predicted to progressively transform into subcluster 1. In subcluster 1, there was a significant upregulation of genes such as BMP and ALPL, signaling enhanced activation of calcification-related pathways. This was highly relevant to end-stage arachnoid ossification formation. After being activated, microglial cells transformed into inflammatory disease-associated microglial cells and continued to express high levels of chemokines CCL2, CCL4, IL-1β, and other inflammatory factors NAMPT, INPP5D and NLRP3. This might be the main reason why AA recurrence is frequently observed in patients. These insights enhance our understanding of the pathological progression of AA and may contribute to the identification of novel therapeutic targets.Copyright © 2025 The Authors. Published by Elsevier B.V. All rights reserved.