Background <p>Soybean is a major oil crop and a primary protein source for livestock, and soybean oil is the most common input for biodiesel. Identifying genes that enhance soybean yield and oil content is crucial for breeding programs. Phosphatidic acid (PA) phosphohydrolase (PAH), which dephosphorylates PA to diacylglycerol (DAG), plays a critical role in lipid synthesis, and yet their potential in improving agronomic traits of oil crops remains unexplored.</p> Results <p>This study shows that seed-specific expression of <i>AtPAH1/2</i> enhances PA turnover into DAG and triacylglycerol (TAG) accumulation in soybean seeds. <i>PAH</i> overexpression upregulated the expression of DAG acyltransferase (<i>DGAT</i>) but suppressed phospholipid: DAG acyltransferase (<i>PDAT</i>). In addition, seed-specific expression of <i>AtPAH1/2</i> increases soybean seed size and weight. Furthermore, analysis of the variation of the soybean PAHs in 4414 soybean accessions indicated that the advantageous effects of <i>GmPAH</i>s on oil content and seed weight were selected during domestication.</p> Conclusion <p>These findings suggest that targeting <i>PAH</i>s represents a promising strategy for enhancing soybean seed oil content and yield in current cultivars and landraces soybeans.</p>