Following human immunodeficiency virus (HIV-1) entry into the host cell, the viral capsid gradually disassembles in a process called uncoating. A proper rate of uncoating is important for reverse transcription of the HIV-1 genome. Host restriction factors such as TRIM5alpha; and TRIMCyp bind retroviral capsids and cause premature disassembly, leading to blocks in reverse transcription. Other host factors, such as cyclophilin A, stabilize the HIV-1 capsid and are required for efficient infection in some cell types. To identify additional cellular factors that alter retroviral core uncoating, we developed a novel in vitro assay of HIV-1 capsid stability. Using this assay, we have shown that a factor in the cytoplasm of cells from multiple vertebrate species slows the spontaneous disassembly of HIV-1 capsid-nucleocapsid (CA-NC) complexes in vitro. We identified the PDZ-Domain-containing protein 8 (PDZD8) as a critical component of the capsid-stabilizing activity in the cytoplasmic extracts. PDZD8 has been previously reported to bind the HIV-1 Gag polyprotein and to make a positive contribution to the efficiency of HIV-1 infection. PDZD8 knockdown accelerated the disassembly of HIV-1 capsids in infected cells, resulting in decreased reverse transcription. The PDZD8 coiled-coil domain is sufficient for HIV-1 capsid binding, but other parts of the protein, including the PDZ domain, are apparently required for stabilizing the capsid and supporting HIV-1 infection. In summary, PDZD8 interacts with and stabilizes the HIV-1 capsid and thus represents a potentially targetable host cofactor for HIV-1 infection.