Senescence-associated β-galactosidase (SA-β-gal) is a widely used biomarker for senescent and aging cells, but its origin and role in senescence are not well understood. This study demonstrates that SA-β-gal activity is derived from the lysosomal β-galactosidase gene (GLB1), which encodes the enzyme typically measured at an acidic pH of 4.5. In fibroblasts from patients with autosomal recessive G934T-gangliosidosis, which lack functional lysosomal β-galactosidase, SA-β-gal activity was not detected even though the cells underwent replicative senescence. Similarly, in normal fibroblasts expressing small-hairpin RNA that depletes GLB1 mRNA, SA-β-gal activity was absent despite the cells undergoing senescence. These findings indicate that SA-β-gal activity is not required for senescence and suggests that it serves as a surrogate marker for increased lysosome number or activity, rather than a specific marker of senescence. The identification of GLB1 as the gene encoding SA-β-gal will facilitate further research on the factors controlling its expression in various growth states.Senescence-associated β-galactosidase (SA-β-gal) is a widely used biomarker for senescent and aging cells, but its origin and role in senescence are not well understood. This study demonstrates that SA-β-gal activity is derived from the lysosomal β-galactosidase gene (GLB1), which encodes the enzyme typically measured at an acidic pH of 4.5. In fibroblasts from patients with autosomal recessive G934T-gangliosidosis, which lack functional lysosomal β-galactosidase, SA-β-gal activity was not detected even though the cells underwent replicative senescence. Similarly, in normal fibroblasts expressing small-hairpin RNA that depletes GLB1 mRNA, SA-β-gal activity was absent despite the cells undergoing senescence. These findings indicate that SA-β-gal activity is not required for senescence and suggests that it serves as a surrogate marker for increased lysosome number or activity, rather than a specific marker of senescence. The identification of GLB1 as the gene encoding SA-β-gal will facilitate further research on the factors controlling its expression in various growth states.