Valproic acid (VPA) is a widely used medication for the treatment of epilepsy, migraines, neuropathic pain, and mood disorders, with contrasting implications across the lifespan. Prenatal exposure is associated with neurodevelopmental risk, whereas postnatal use has established therapeutic benefits. This systematic review aimed to synthesize pathway-oriented mechanistic evidence on biological pathways affected by VPA across in vivo, in vitro, in silico, and clinical studies. A comprehensive search was conducted in Medline/PubMed, Web of Science, Scopus, and Embase from January 2009 to June 2026. Records were screened in duplicate, and study-level data were extracted on exposure window and dose, experimental model or tissue, and pathway-level outcomes. Risk of bias was assessed qualitatively in eligible studies using study-design specific criteria informed by SYRCLE and ROBINS-I tools. Due to substantial heterogeneity across study designs, experimental models, exposure windows, doses, and molecular outcomes, findings were synthesized narratively within a systematic review framework. A total of 385 studies met eligibility criteria. VPA was reported to modulate embryonic developmental processes, oxidative stress, gene and protein expression, epigenetic regulation, immune function, cellular energy metabolism, cytotoxicity and apoptosis, ERK/MAPK and PI3K/AKT/mTOR-related signaling, neurotransmitter systems, and oncological pathways. Across models, the exposure window emerged as a critical determinant of biological impact. The review protocol was registered in PROSPERO (CRD420261351346). By integrating pathway-oriented evidence across heterogeneous models, this review contributes to a more critical understanding of VPA-induced biological mechanisms and highlights mechanistic domains that warrant future experimental and translational research.