Microtubules (MTs) are key components of the eukaryotic cytoskeleton, formed by dynamic heterodimers of α- and β-tubulin subunits that constantly assemble and disassemble within cells. Research into microtubule dynamics is crucial due to its relevance in anticancer therapies. In mammals, various β-tubulin subtypes exist, with βIII-tubulin (TUBB3) recognized as a significant neuronal biomarker and linked to poor prognosis in cancer patients due to its high expression in tumors. Despite advances in chemotherapy, drug resistance—often attributed to elevated TUBB3 levels—poses significant challenges to effective treatment. Although role of TUBB3 in nervous system is acknowledged, its precise functions and mechanisms in relation to cancer therapies are not fully understood. This review offers an overview of TUBB3’s biological function, its distinctions from other β-tubulin isoforms, examines its role in cancer, elucidates its mechanisms of action, and evaluates therapeutic strategies aimed at targeting TUBB3 to combat drug resistance in tumors.