Global increase of life expectancy is rarely accompanied by increased health span, calling for a greater understanding of age-associated behavioral decline. Motor independence is strongly associated with the quality of life of elderly people, yet the regulators for motor aging have not been systematically explored. Here, we designed a fast and efficient genome-wide screening assay inCaenorhabditis elegansand identified 34 consistent genes as potential regulators of motor aging. Among the top hits, we found VPS-34, the class III phosphatidylinositol 3-kinase that phosphorylates phosphatidylinositol (PI) to phosphatidylinositol 3-phosphate (PI(3)P), regulates motor function in aged but not young worms. It primarily functions in aged motor neurons by inhibiting PI(3)P-PI-PI(4)P conversion to reduce neurotransmission at the neuromuscular junction (NMJ). Genetic and pharmacological inhibition of VPS-34 improve neurotransmission and muscle integrity, ameliorating motor aging in both worms and mice. Thus, our genome-wide screening revealed an evolutionarily conserved, actionable target to delay motor aging and prolong health span.