HIV-1 replication has been intensively investigated over the past 30 years. Hsp90 is one of the most abundant proteins in human cells, important in the formation and function of several protein complexes that maintain cell homeostasis. Remarkably, the impact of Hsp90 on HIV-1 infection has started to be appreciated only recently. Hsp90 has been shown to (a) promote HIV-1 gene expression in acutely infected cells, (b) localize at the viral promoter DNA, (c) mediate enhanced replication in conditions of hyperthermia and (d) activate the P-TEFb complex, which is essential for efficient HIV-1 transcription. Hsp90 has been implicated in buffering deleterious mutations of the viral core and in the regulation of innate and acquired immune responses to HIV-1 infection. Therefore, Hsp90 is an important host factor promoting several steps of the HIV-1 life cycle. Several small Hsp90 inhibitors are in Phase II clinical trials for human cancers and might potentially be used to inhibit HIV-1 infection at multiple levels.