Growing evidence suggests that iron accumulation in the substantia nigra (SN) plays an important role in the degeneration of dopamine neurons in Parkinson’s disease (PD). Slowing down the accumulation of iron in cells have been shown to be the key point for delaying and treating PD. Myricetin has been reported to have the biological functions of anti-oxidation, anti-apoptosis, anti-inflammation and iron-chelation. The aim of the present study is to investigate the neuroprotective effect of myricetin on rotenone-treated MES23.5 cells and the underlying mechanisms. The results showed that myricetin significantly alleviates rotenone-induced decrease in the cell viability. Myricetin also suppresses the production of intracellular reactive oxygen species (ROS) and restores the mitochondrial transmembrane potential (Δψm) that was induced by rotenone. In addition, myricetin significantly suppresses rotenone-induced hepcidin gene transcription and partly relieves rotenone-induced inhibition in Ferroportin 1 (Fpn 1) expression both in mRNA and protein levels. Furthermore, myricetin also inhibits rotenone-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3) and mothers against decapentaplegic homolog 1 (SMAD1) in the MES23.5 cells. We conclude that myricetin protects rotenone-treated MES23.5 cells by preventing against iron accumulation via potently inhibiting hepcidin expression by altering STAT3 and SMAD1 signaling pathways.