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Abstract Recently, the presence of different nanoparticles (NPs) has developed targeting drug delivery in treatment of cancer cell. Targeted drug delivery systems using NPs have shown great promise in improving the efficacy of intracellular uptake as well as local concentration of therapeutics with minimizing side effects. The current study planned to synthesized resveratrol-loaded magnetic niosomes nanoparticles (RSV-MNIONPs) and evaluate their cytotoxicity activity in pancreatic cancer cells. For this aim, magnetic nanoparticles (MNPs) were synthesized and loaded into niosomes (NIOs) by the thin film hydration technique and then characterized via DLS, FT-IR, TEM, SEM and VSM techniques. Moreover, the cytotoxic activity of the RSV-MNIONPs on the Capan-1 cells line was assessed by the MTT test. The distribution number of RSV-MNIONPs was gained about 80 nm and 95 nm with surface charge of − 14.0 mV by SEM and TEM analysis, respectively. RSV loading efficacy in NIOs was about 85%, and the drug releases pattern displayed a sustained discharge with a maximum amount about 35% and 40%, within 4 h in pH = 7.4 and pH = 5.8, respectively. The cytotoxicity of the RSV-MNIONPs in the presence of an external magnetic field is higher than that of the RSV, indicating enhanced cellular uptake in their encapsulated states. Furthermore, RSV loaded MNNPs were found to induce more cell cycle arrest at the G0/G1 checkpoint than free RSV. Compared with RSV-treated cells, the mRNA expression levels of BAX, Bcl2, FAS, P 53, Cyclin D and hTERT, were significantly changed in cells treated with RSV loaded MNNPs. The niosomes NPs approaches have been widely used to attain higher solubility, improved bioavailability, enhanced stability, and control delivery of RSV. Our formulation displayed antitumor activity and can be considered an appropriate carrier with a great potential for future usage in cancer therapy.