Cellular senescence contributes to accelerated aging, neuroinflammation, and the development of HIV-associated neurocognitive disorders (HAND) in the era of combined antiretroviral therapy (cART). One HIV viral factor that could lead to cellular senescence is the persistence of HIV-1 Tat in the brain. As a secreted viral protein, Tat is known to enter endolysosomes of cells through receptor-mediated endocytosis, and we have shown that Tat induces endolysosome damage and dysfunction. Significantly, endolysosome dysfunction has been strongly linked to cellular senescence. However, it is not known whether endolysosome dysfunction represents a driver or consequence of cellular senescence. Because Tat-induced endolysosome damage represents an early step in exogenous Tat-induced cellular senescence, we tested the hypothesis that Tat induces cellular senescence via an endolysosome-dependent mechanism in human astrocytes. We demonstrated that Tat interacts with an endolysosome-resident Toll-like receptor 7 (TLR7) via its arginine-rich basic domain, and such an interaction results in endolysosome damage and the development of a senescence-like phenotype including cell cycle arrest, enhanced SA-β-gal activity, and increased release of senescence-associated secretory phenotype (SASP) factors (IL-6, IL-8, and CCL2). Thus, our finding provided mechanistic insights whereby Tat induces endolysosome damage and cellular senescence in human astrocytes. We provide compelling evidence that endolysosome damage drives the development of cellular senescence. Our findings also highlight the novel role of TLR7 in the development of cellular senescence and suggest that TLR7 represents a novel therapeutic target against senescence and the development of HAND.