The present study proposes a novel mode of action for cyclophilin A (CypA) in the HIV-1 life cycle. We demonstrate that CypA-deficient viruses do not replicate because they fail to attach to target cells. We show that CypA is exposed at the viral membrane and mediates HIV-1 attachment. We identify heparan as the exclusive cellular binding partner for CypA. Furthermore, CypA binds directly to heparan via a domain rich in basic residues similar to known heparin-binding motifs. This interaction between exposed CypA and cell surface heparans represents the initial step of HIV-1 attachment and is a necessary precursor to gp120-binding to CD4. In conclusion, HIV-1 attachment to target cells is a multi-step process that requires an initial CypA-heparan interaction followed by the gp120-CD4 interaction.