A large body of evidence suggests that cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT), as well as expression and function of retinoid receptors, are pivotal features of tumor initiation, progression, and chemoresistance. This is also true for pancreatic ductal adenocarcinoma (PDAC), which represents a clinical challenge due to poor prognosis and increasing incidence. Understanding the above features of cancer cells could open new avenues for PDAC treatment strategies. The aim of this study was to investigate the relation between CSCs, EMT, and retinoid receptors in PDAC after treatment with the chemotherapeutic agents - gemcitabine and 5-fluorouracil. First, we demonstrated the difference in the expression levels of CSC and EMT markers and retinoid receptors in the untreated Mia PaCa-2 and Panc1 cells that also differed in the frequency of spontaneous apoptosis and distribution between the cell cycle phases. Chemotherapy reduced the number of cancer cells in the S phase. Gemcitabine and 5-fluorouracil modulated expression of CSC markers, E-cadherin, and RXRβ in Panc1 but not in Mia PaCa-2 cells. We suggest that these effects could be attributed to the difference in the basal levels of expression of the investigated genes. The obtained data could be interesting in the context of future preclinical research.