The mitochondrial serine β-lactamase-like protein LACTB has emerged as a critical regulator in cancer biology, distinguished by its unique structural and functional attributes. Defined by its conserved penicillin-binding proteins and β-lactamases (PBP-βLs) domain and SXXK catalytic motif, LACTB demonstrates properties distinct from its prokaryotic homologs, including the ability to polymerize into filaments. These structural characteristics enable LACTB to modulate mitochondrial organization and enzymatic activity, influencing lipid metabolism and indirectly affecting cellular proliferation. Importantly, the expression and functional roles of LACTB exhibit cancer-type-specific variation, underscoring its dual function as both a tumor suppressor and an oncogene. Decreased LACTB expression is associated with poor clinical outcomes in cancers such as breast cancer, lung cancer, and colorectal cancer, while specific mutations and regulatory mechanisms have been linked to its oncogenic activity in osteosarcoma and pancreatic adenocarcinoma. Mechanistically, LACTB regulates key processes in cancer progression, including mitochondrial dynamics, epithelial-mesenchymal transition (EMT), and cell death pathways. This duality highlights LACTB as a promising therapeutic target and underscores its relevance in advancing precision oncology strategies. This review provides a comprehensive analysis of expression level, structure-function relationships, and the diverse roles of LACTB in oncogenesis, underscoring its promise as a focal point for precision cancer therapies.