HIV-1 Tat is a key viral protein that stimulates several steps of viral gene expression. Tat is especially required for the transcription of viral genes. Nevertheless, it is still not clear if and how Tat is incorporated into HIV-1 virions. Cyclophilin A (CypA) is a prolyl isomerase that binds to HIV-1 capsid protein (CA) and is thereby encapsidated at the level of 200 to 250 copies of CypA/virion. Here, we found that a Tat-CypA-CA tripartite complex assembles in HIV-1-infected cells and allows Tat encapsidation into HIV virions (1 Tat/1 CypA). Biochemical and biophysical studies showed that high-affinity interactions drive the assembly of the Tat-CypA-CA complex that could be purified by size exclusion chromatography. We prepared different types of viruses devoid of transcriptionally active Tat. They showed a 5- to 10 fold decrease in HIV infectivity, and conversely, encapsidating Tat into ΔTat viruses greatly enhanced infectivity. The absence of encapsidated Tat decreased the efficiency of reverse transcription by ~50% and transcription by more than 90%. We thus identified a Tat-CypA-CA complex that enables Tat encapsidation and showed that encapsidated Tat is required to initiate robust viral transcription and thus viral production at the beginning of cell infection, before neosynthesized Tat becomes available. IMPORTANCE The viral transactivating protein Tat has been shown to stimulate several steps of HIV gene expression. It was found to facilitate reverse transcription. Moreover, Tat is strictly required for the transcription of viral genes. Although the presence of Tat within HIV virions would undoubtedly favor these steps and therefore enable the incoming virus to boost initial viral production, whether and how Tat is present within virions has been a matter a debate. We here described and characterized a tripartite complex between Tat, HIV capsid protein, and the cellular chaperone cyclophilin A that enables efficient and specific Tat encapsidation within HIV virions. We further showed that Tat encapsidation is required for the virus to efficiently initiate infection and viral production. This effect is mainly due to the transcriptional activity of Tat.