α-Linolenic acid (ALA) is an essential fatty acid crucial for improving dietary nutrition in humans. Since ALA can only be obtained from dietary sources, developing ALA-rich biological resources is imperative. The seed oil of tree peony (Paeonia × suffruticosa), a promising woody oil crop, is particularly rich in ALA. In this study, we identified PsbZIP44, a basic leucine zipper (bZIP) transcription factor in tree peony, as a key regulator of ALA accumulation in seeds. Heterologous expression of PsbZIP44 in Arabidopsis (Arabidopsis thaliana) promoted ALA accumulation, and transient silencing in tree peony seeds decreased ALA levels. Additionally, we found that another bZIP transcription factor, PsbZIP9, negatively regulates ALA biosynthesis by inhibiting PsbZIP44 activity during the late stages of seed development. Based on these findings, we propose a model for FATTY ACID DESATURASE3 (PsFAD3) regulation in tree peony seeds, in which PsbZIP44 activates PsFAD3 during early seed development to promote ALA biosynthesis, while PsbZIP9 suppresses PsbZIP44 activity in later stages, limiting ALA accumulation. This study highlights the synergistic role of transcription factors in seed development, reveals the regulatory mechanisms of PsFAD3, and enhances our understanding of ALA biosynthesis. These findings provide a foundation for improving the quality of tree peony seed oil.