Among rare earth elements, cerium has the unique ability of regulating the growth of plant cells and the biosynthesis of metabolites at different stages of plant development. The signal pathways of Ce3+-mediated ginsenosides biosynthesis in ginseng hairy roots were investigated. At a low concentration, Ce3+ improved the elongation and biomass of hairy roots. The Ce3+-induced accumulation of ginsenosides showed a high correlation with the reactive oxygen species (ROS), as well as the biosynthesis of endogenous methyl jasmonate (MeJA) and ginsenoside key enzyme genes (PgSS, PgSE and PgDDS). At a Ce3+ concentration of 20 mg Lâˆ`1, the total ginsenoside content was 1.7-fold, and the total ginsenosides yield was 2.7-fold that of the control. Malondialdehyde (MDA) content and the ROS production rate were significantly higher than those of the control. The activity of superoxide dismutase (SOD) was significantly activated within the Ce3+ concentration range of 10 to 30 mg Lâˆ`1. The activity of catalase (CAT) and peroxidase (POD) strengthened with the increasing concentration of Ce3+ in the range of 20â€“40 mg Lâˆ`1. The Ce3+ exposure induced transient production of superoxide anion (O2â€¢âˆ`) and hydrogen peroxide (H2O2). Together with the increase in the intracellular MeJA level and enzyme activity for lipoxygenase (LOX), there was an increase in the gene expression level of MeJA biosynthesis including PgLOX, PgAOS and PgJMT. Our results also revealed that Ce3+ did not directly influence PgSS, PgSE and PgDDS activity. We speculated that Ce3+-induced ROS production could enhance the accumulation of ginsenosides in ginseng hairy roots via the direct stimulation of enzyme genes for MeJA biosynthesis. This study demonstrates a potential approach for understanding and improving ginsenoside biosynthesis that is regulated by Ce3+-mediated signal transduction.