【Objective】 To investigate the effect of prenatal stress (PS) on the profile changes of cardiac metabolites in offspring rats and to analyze the potential role of key differentiators in key differential signaling pathways. 【Methods】 UHPLC-Q-TOF/MS analysis was used to detect the changes of metabolite profile in the heart tissues of offspring rats in control group and PS group. KEGG pathway annotation and analysis were used to screen out metabolic differences in key signaling pathways and quantify their expression levels so as to predict the potential function of these key molecules in the effect of PS on the heart tissues of offspring rats. 【Results】 Compared with the control group rats, the signaling pathways in the PS offspring rats’ heart tissue that changed significantly included biosynthesis of amino acids, purine metabolism, pyrimidine metabolism, alanine, aspartate and glutamate metabolism, cAMP signaling pathways, arginine biosynthesis, GABAergic synapses, glutamate synapse, nicotine addiction, and regulation of actin cytoskeleton. Among them, the levels of L-glutamine, pseuduracil, uric acid, xanthine, 2’-deoxyadenosine 5’-monophosphate, cytosine 3’-monophosphate, and cytosine 5’-monophosphate were upregulated, while the level of argininosuccinic acid was downregulated, which enriched in purine metabolism, pyrimidine metabolism, and arginine biosynthesis pathway. 【Conclusion】 PS leads to abnormal changes of L-glutamine, pseuduracil, uric acid, and xanthine in the heart tissue of offspring rats, and PS may be a high risk factor for cardiovascular diseases in offspring rats.