Pancreatic ductal adenocarcinoma (PDAC) is highly malignant and has a poor prognosis, without effective therapeutic targets in common gene mutations. Gemcitabine, a first-line chemotherapeutic for PDAC, confers <10 % 5-year survival rate because of drug resistance. Y-box binding protein 1 (YBX1), associated with multidrug-resistance gene activation, remains unelucidated in PDAC gemcitabine resistance. In vivo and in vitro, we verified YBX1's promotional effects, especially gemcitabine resistance, in pancreatic cancer cells. YBX1-induced LRP1 transcription by binding to the LRP1 promoter region significantly altered the concentration and distribution of β-catenin in pancreatic cancer cells. Through TCF3, β-catenin bound to the promoter region of RRM1, a key gene for gemcitabine resistance, that promotes RRM1 expression. Combination therapy with the YBX1 inhibitor SU056 and gemcitabine effectively reduced gemcitabine resistance in in vivo and in vitro experiments. High YBX1 expression promoted pathogenesis and gemcitabine resistance in pancreatic cancer through the YBX1-LRP1-β-catenin-RRM1 axis. Combining YBX1 inhibitors with gemcitabine may provide a new direction for combination chemotherapy to overcome gemcitabine resistance, which frequently occurs during chemotherapy for pancreatic cancer.