BACKGROUND: Maternal antenatal corticosteroid treatment is standard care to accelerate fetal maturation. However, there are growing concerns that antenatal corticosteroid administration may harm fetal neurodevelopment. Quantitative assessments of the effects of antenatal corticosteroid on the neonates have not been performed and poorly understood about their complex biology. RESULTS: We collected Methylation BeadChips-generated DNA methylation data from the Gene Expression Omnibus (GEO) database and then employed "multi-tissue predictor" to quantify the DNAm age of saliva from 36 preterm neonates, which were stratified by the absence (n = 12) or presence (n = 24) of antenatal corticosteroid exposure, as well as 36 full-term neonates. Next, the DNAm age of human lung fibroblast IMR90 cells and human fetal multipotent hippocampal progenitor HPC cells, with or without glucocorticoid treatment, was also determined. We observed that the DNAm age of full-term neonates was significantly higher than that of the preterm neonates, and antenatal corticosteroid exposure accelerated the DNAm age of preterm neonates, while glucocorticoid exposure accelerated the DNAm age of IMR90 cells. Conversely, dexamethasone exposure delayed the DNAm age of HPC cells during the proliferation phase. It is noteworthy that 65% of the differentially methylated probes (DMPs) linked to the multi-tissue predictor marked CpGs and corticosteroid exposure in IMR90 cells exhibited comparable methylation patterns with the DMPs associated with the antenatal corticosteroid exposure in preterm neonates. Conversely, the majority of these DMPs exhibited inverse methylation alterations in dexamethasone-induced HPC cells. Furthermore, the epigenome-wide association study (EWAS) trait enrichment analyses of the DMPs linked to the antenatal corticosteroid exposure in preterm neonates revealed significant associations with prenatal adverse environmental exposure, growth and development, and neuropsychiatric disorders. CONCLUSIONS: Our results identified the cellular and molecular evidences of epigenetic clock changes in neonatal growth and developmental trajectories with the interference of antenatal corticosteroid treatment and provided potential clinical guidance for the future development of noninvasive fetal assessments to identify pregnant women who could benefit from antenatal corticosteroid in a wider gestational age.