Pancreatic stellate cells (PSCs) contribute to pancreatic ductal adenocarcinoma (PDAC) progression and therapeutic resistance, yet their detailed functions remain unclear. This study combined RNA sequencing and assay for transposase-accessible chromatin using sequencing (ATAC-seq) on sorted PSCs from adjacent normal and PDAC tissues to investigate their transcriptional and epigenetic activation. PSCs heterogeneity and functions are characterized through bulk, single-cell, and spatial transcriptomes, as well as in situ sequencing. The clinical relevance of PSCs in immunotherapy is assessed using an in-house immune-checkpoint blockade (ICB) treatment cohort. Findings showed that stress and hypoxia signaling activated PSCs in PDAC. Three common PSCs (CPSCs) and four tumor-associated PSCs (TPSCs) are identified, each with distinct functions. CPSCs differentiated into CCL19+ TPSCs in immune-enriched regions, MYH11+ TPSCs in the stromal region, and PLXDC1+ TPSCs, which exhibited cancer-associated myofibroblasts (myCAFs) phenotype linked to poor prognosis. Notably, PLXDC1+ TPSCs, located near aggressive LRRC15+ myCAFs and SPP1+ macrophages, formed a desmoplastic and immunosuppressive niche around the tumor boundary, promoting CD8 T cell exhaustion. Single-cell transcriptomics of PDAC patients treated with ICB revealed that PLXDC1+ TPSCs correlated with poor immunotherapy efficacy. Overall, this study provides key insights into PSCs in PDAC and potential therapeutic targets.