Chemotherapy resistance contributes to the unsatisfied prognosis in pancreatic cancer (PC) patients. Heat shock protein beta-1 (HSPB1) plays a tumor promoting role in PC by inhibiting ferroptosis. This study aims to explore whether high expression of HSPB1 was responsible for ferroptosis and gemcitabine (GEM) resistance in PC. Here, we found that HSPB1 was upregulated in GEM-resistant PC cells and tumor tissues, as confirmed by RT-qPCR and Western blotting assays. Knockdown of HSPB1 enhanced GEM sensitivity, decreased the abilities of proliferation and invasion, and promoted apoptosis in GEM-resistant PC cells. Utilizing commercial kits, HSPB1 inhibition triggered ferroptosis, as indicated by increased levels of reactive oxygen species, malondialdehyde, and Fe, along with reduced glutathione (GSH) levels. Furthermore, the methylation specific PCR (MSP) results demonstrated a significant decrease in the methylation level of annexin A2 (ANXA2) CpG. The Chromatin immunoprecipitation (ChIP), ChIP-Re-ChIP, and Co-IP experiments revealed that HSPB1 interacts with lysine-specific histone demethylase 1A (KDM1A), recruiting KDM1A-CoREST complex to the ANXA2 promoter to enhance ANXA2 expression through demethylation of H3K9me2. Additionally, ANXA2 depletion further inhibited cell proliferation and invasion and induced ferroptosis in KDM1A-silenced cells, whereas ANXA2 overexpression produced the opposite effects. Finally, HSPB1 overexpression reduced gemcitabine sensitivity by promoting tumor growth in nude mice. Altogether, HSPB1 promoted ANXA2 expression by facilitating H3K9me2 demethylation through the recruitment of KDM1A-CoREST complex to the ANXA2 promoter, thereby inhibiting ferroptosis and enhancing GEM resistance in PC. These data provided a new insight for overcoming GEM-resistant PC.