<p>Lactate has long been viewed as a “waste product” of anaerobic glycolysis, with its role in health and disease often overlooked. However, recent discoveries of lactylation—a novel post-translational modification involving lactate—have sparked a renewed understanding of lactate’s functions. Lactylation alters the molecular structure of proteins with different cellular localizations, enabling the regulation of their functions and aggregation in specific spatiotemporal contexts, with its impact on biomolecular phase separation being one of its primary effects. However, it remains unknown how lactylation dynamically regulates the spatiotemporal specificity of phase separation and its role in diseases. This article provides an overview of the regulatory mechanisms of biomolecular phase separation driven by lactylation, aiming to offer fresh insights into the role of lactylation in normal and disease-related biological processes while deepening our understanding of its research value and biological significance.</p>