Although gemcitabine-based chemotherapy is common and effective for pancreatic cancer (PC), acquired drug resistance is one of the major reasons for treatment failure. Therefore, a novel therapeutic approach for gemcitabine-resistant PC is required. Nuclear factor erythroid 2-related factor 2 (Nrf2) is an oxidative stress-responsive transcription factor regulating antioxidant responses and plays a crucial role in chemoresistance. In the present study, the antitumor activity of periplocin, a natural cardiac glycoside, was evaluated in an established gemcitabine-resistant PC cell line (PANC-GR). Nrf2 was overexpressed in gemcitabine-resistant cells, and Nrf2 knockdown recovered gemcitabine sensitivity in PANC-GR cells. The antiproliferative activity of periplocin was highly associated with Nrf2 downregulation and Nrf2-mediated signaling pathways in PANC-GR cells. Periplocin also increased reactive oxygen species production inducing G₀/G₁ cell cycle arrest and apoptosis in PANC-GR cells. Periplocin and gemcitabine combined significantly inhibited tumor growth in a PANC-GR cells-implanted xenograft mouse model via Nrf2 downregulation. Overall, these findings suggest that periplocin might be a novel therapeutic agent against gemcitabine resistance, as it could recover sensitivity to gemcitabine by regulating Nrf2-mediated signaling pathways in gemcitabine-resistant PC cells.