IntroductionPrenatal stress (PS) can cause depression in offspring. However, the underlying biological mechanism of these influences is still unclear. This work was implemented to investigate the molecular mechanisms of depressive‐like behavior of offspring rats insulted with PS.MethodsRelative quantitative phosphoproteomics of the hippocampus of PS susceptibility (PS‐S) and control (CON) rat offspring was performed using liquid chromatography‐tandem mass spectrometry to confirm known pathways and to identify new mechanisms involved in depression.ResultsA total of 6790 phosphopeptides, 9817 phosphorylation sites, and 2978 phosphoproteins were detected. Among the 2978 phosphoproteins, 1760 (59.09%) had more than two phosphorylated sites, the ENSRNOP00000023460 protein had more than 117 phosphorylated sites, and the average distribution of modification sites per 100 amino acids was 2.97. There were 197 different phosphopeptides, including 140 increased phosphopeptides and 57 decreased phosphopeptides in the PS‐S offspring rats, compared to the CON offspring rats. These differential phosphopeptides corresponded to 100 upregulated and 44 downregulated phosphoproteins, respectively. Gene ontology enrichment analysis revealed that these different phosphoproteins in the top five enriched terms in the cellular component, molecular function, and biological proces categories were involved in a total of 35 different phosphoproteins, and these phosphoproteins were mainly related to myelin‐, microtubule‐ and synapse‐associated proteins. The enrichment of Kyoto Encyclopedia of Genes and Genome pathways was found to be involved in many essential biological pathways, and the top five pathways included amphetamine addiction, insulin secretion, Cushing syndrome, and the circadian entrainment signaling pathway. These first five pathways were related to nine phosphoproteins, including Adcy9, Apc, Cacna1c, Camk2a, Camk2b, Camk2g, Ctnnd2, Grin2a, and Stx1a. The full data are available via ProteomeXchange with identifier PXD019117.ConclusionWe preliminarily identified 144 different phosphoproteins involved in myelin, microtubule, and synapse formation and plasticity in the hippocampus of susceptible offspring rats exposed to PS.