Plasminogen activator inhibitor (PAI-1) expression has been associated with a higher risk of development of obesity. DNA methylation (DNAm) is an epigenetic mechanism regulating gene transcription and likely involved in the fetal programming of childhood obesity. Our study aimed to assess the associations between PAI-1 gene (SERPINE1) DNAm, plasma PAI-1 levels, and adiposity at five years of age. We analyzed DNAm and anthropometric data from 146 girls and 177 boys from the Gen3G prospective birth cohort. We assessed adiposity using BMI z-scores, waist circumference, total skinfolds, and percentages of total, android, and trunk fat measured by dual-energy radiography (DXA). We estimated blood cell DNAm levels at 15 CpG sites within SERPINE1 using the methylationEPIC array. After correction for multiple testing, we found that lower DNAm in SERPINE1 intron 3 (cg11353706) was associated with greater adiposity levels in girls (waist circumference: r = −0.258, p = 0.002; skinfolds: r = −0.212, p = 0. 013; android fat: r = −0.215, p = 0.015; BMI z-score: r = −0.278, p < 0.001) and that lower DNAm in the SERPINE1 promoter (cg19722814) was associated with higher plasma PAI-1 levels in boys (r = −0.178, p = 0.021). Our study suggests that DNAm levels at the SERPINE1 gene locus are negatively correlated with adiposity, but not with plasma PAI-1 levels, in young girls only.