We have previously reported that morphine induces apoptosis. However, the underlying molecular mechanisms remain to be elucidated. Toll-like receptor 2 (TLR2), a key immune receptor in the TLR family, modulates cell survival and cell death in various systems. Evidence indicates that beta-arrestin 2 acts as a negative regulator of innate immune activation by TLRs. Here, we investigated the roles of TLR2, the downstreaming mediator MyD88, and beta-arrestin 2 in morphine-induced apoptosis. We showed that overexpression of TLR2 in HEK293 cells caused a significant increase in apoptosis after morphine treatment. Inhibition of MyD88 by transfecting dominant negative MyD88 or overexpression of beta-arrestin 2 by transfecting beta-arrestin 2 full length plasmid in TLR2 overexpressing HEK293 cells attenuated morphine-induced apoptosis. Our study thus demonstrates that TLR2 signaling mediates the morphine-induced apoptosis, and beta-arrestin 2 is a negative regulator in morphine-induced, TLR2-mediated apoptosis.