The uncoating of HIV-1 cores released into the cytoplasm as a result of viral fusion is a critical step en route to productive infection. The sites, the timing, and the extent of uncoating, defined as shedding of the capsid protein (CA) from the core complex encasing the viral genome, remain poorly understood. To elucidate this elusive step of HIV-1 entry, we developed a novel strategy to visualize the CA loss using a fluorescently tagged oligomeric form of cyclophilin A (CypA-DsRed), which binds CA with high affinity. CypA-DsRed is specifically packaged into virions and remains associated with cores after permeabilization of the viral membrane. Importantly, we show that CypA-DsRed and CA are lost concomitantly from the cores in vitro and in living cells. The rate of CypA-DsRed loss is modulated by mutations that alter the core stability and is accelerated by reverse transcription. Whereas the majority of single cores loose CypA-DsRed shortly after viral fusion, a small fraction remains intact for several hours. Single particle tracking at late times post-infection reveals a gradual loss of CypA-DsRed which is blocked upon inhibition of reverse transcription. These late uncoating events occur both in the cytoplasm and after docking at the nuclear membrane. In conclusion, the CypA-DsRed-based imaging assay enables time-resolved visualization of single HIV-1 uncoating in living cells. This novel approach provides important clues regarding spatio-temporal regulation of uncoating. This work was supported by the NIH R01 GM054787 grant and Pittsburgh Center for HIV Protein Interactions (P50GM082251).