Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with poor prognosis and limited therapeutic options. In a previous publication, our group defined some of the mechanisms that vitamin D analogue paricalcitol (P) and hydroxychloroquine (H) potentiated the effects of gemcitabine-based chemotherapy in PDAC. Based on this, we hypothesized that PH may potentiate 5-fluorouracil (5FU) and oxaliplatin-based chemotherapy, and this may involve a novel mechanism of extracellular matrix (ECM) modulation. The combination of PH with 5FU/oxaliplatin significantly increased the cell death, apoptosis, and S-phase cell cycle arrest as compared to untreated or 5FU/oxaliplatin treated MIA PaCa-2, HPAC and KPC cell lines. In vivo, the combination therapy inhibited PDAC growth and altered the immune landscape by activating T and NK cells. Proteomic analysis revealed significant reduction in ECM proteins, specifically integrin beta-4 (ITGB4). Confirmation of the role of ITGB4 was performed through genetic knockdown of ITGB4 which led ECM inhibition. In conclusion, the combination of PH significantly enhances the efficacy of oxaliplatin and 5FU. We identified a new mechanism of action of PH through inhibiting ITGB4 leading to ECM modulation. These results suggest that the combination of PH with cytotoxic chemotherapy should be tested in PDAC clinical trials.