As a promising energy plant for biodiesel, Jatropha curcas is a tropical and subtropical shrub and its growth is affected by one of major abiotic stress, chilling. Therefore, we adopt the phosphoproteomic analysis, physiological measurement and ultrastructure observation to illustrate the responsive mechanism of J. curcasseedling under chilling (4 °C) stress. After chilling for 6 h, 308 significantly changed phosphoproteins were detected. Prolonged the chilling treatment for 24 h, obvious physiological injury can be observed and a total of 332 phosphoproteins were examined to be significantly changed. After recovery (28 °C) for 24 h, 291 phosphoproteins were varied at the phosphorylation level. GO analysis showed that significantly changed phosphoproteins were mainly responsible for cellular protein modification process, transport, cellular component organization and signal transduction at the chilling and recovery periods. On the basis of protein-protein interaction network analysis, phosphorylation of several protein kinases, such as SnRK2, MEKK1, EDR1, CDPK, EIN2, EIN4, PI4K and 14-3-3 were possibly responsible for cross-talk between ABA, Ca²⁺, ethylene and phosphoinositide mediated signaling pathways. We also highlighted the phosphorylation of HOS1, APX and PIP2 might be associated with response to chilling stress in J. curcas seedling. These results will be valuable for further study from the molecular breeding perspective.