Proline is one of the most important osmoregulatory solutes subjected to osmotic stresses. In this study, low nitrogen supply suppressed the dry biomass, leaf area, and proline biosynthesis of the seedlings of the energy plant Jatropha curcas, which could grow in poor, dry soil. Low-nitrogen stress induced JcP5CS mRNA expression and decreased the activity of P5CS enzyme and the content of free proline in leaves of J. curcas seedlings. When the seedlings grown in low-nitrogen conditions were suddenly exposed to PEG-6000 (-1.6 MPa) stress, the expression of JcP5CS gene was highly induced, and both the activity of P5CS and the content of free proline increased and maintained at high levels to mitigate the impact of drought stresses. This may be one of the reasons why J. curcas could adapt to poor and drought conditions.