Osteoarthritis (OA) is a whole-joint disease in which the role of the infrapatellar fat pad (IFP) in its pathogenesis is unclear. Our study explored the cellular heterogeneity of IFP to understand OA and identify therapeutic targets.Single-cell and single-nuclei RNA sequencing were used to analyze 10 IFP samples, comprising 5 from OA patients and 5 from healthy controls. Analyses included differential gene expression, enrichment, pseudotime trajectory, and cellular communication, along with comparative studies with visceral and subcutaneous fats. Key subcluster and pathways were validated using multiplex immunohistochemistry.The scRNA-seq performed on the IFPs of the OA and control group profiled the gene expressions of over 49,674 cells belonging to 11 major cell types. We discovered that adipose stem and progenitor cells (ASPCs), contributing to the formation of both adipocytes and synovial-lining fibroblasts (SLF). Interstitial inflammatory fibroblasts (iiFBs) were a subcluster of ASPCs that exhibit notable pro-inflammatory and proliferative characteristics. We identified four adipocyte subtypes, with one subtype showing a reduced lipid synthesis ability. Furthermore, iiFBs modulated the activities of macrophages and T cells in the IFP. Compared to subcutaneous and visceral adipose tissues, iiFBs represented a distinctive subpopulation of ASPCs in IFP that regulated cartilage proliferation through the MK pathway.This study presents a comprehensive single-cell transcriptomic atlas of IFP, uncovering its complex cellular landscape and potential impact on OA progression. Our findings highlight the role of iiFBs in OA, especially through MK pathway, opening new avenues for understanding OA pathogenesis and developing novel targeted therapies.Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.