Cell-associated HIV-1 infection has been proposed to play a pivotal role in the spread of HIV-1 infection. Granulocytes are a category of white blood cells, comprising mainly basophils, neutrophils, and eosinophils, and participate in various inflammatory reactions and defense against pathogens. Here, we investigated the role of human blood granulocytes in the dissemination of HIV-1. These cells were found to express a variety of HIV-1 attachment factors (HAFs). Basophils expressed HAFs dendritic cell (DC)-specific intercellular adhesion molecule 3 (ICAM3)-grabbing nonintegrin (DC-SIGN), DC immunoreceptor (DCIR), heparan sulfate proteoglycan (HSPG), and alpha 4 beta 7 integrin and mediated the most efficient capture of HIV-1 on the cell surface. Neutrophils were found to express DCIR and demonstrated limited efficiency of viral capture. Eosinophils expressed alpha 4 beta 7 integrin but exhibited little or no virus-binding capacity. Intriguingly, following direct contact with CD4(+) T cells, viruses harbored on the surface of basophils were transferred to T cells. The contact between basophils and CD4(+) T cells and formation of infectious synapses appeared necessary for efficient HIV-1 spread. In HIV-1-infected individuals, the frequency of basophils remained fairly stable over the course of disease, regardless of CD4(+) T depletion or the emergence of AIDS-associated opportunistic infections. Collectively, our results provide novel insights into the roles of granulocytes, particularly basophils, in HIV-1 dissemination. Thus, strategies designed to prevent basophil-mediated viral capture and transfer may be developed into a new form of therapy. IMPORTANCE Cell-associated HIV-1 infection has been proposed to play a pivotal role in the spread of HIV-1 infection. Here, we demonstrated that human blood-circulating granulocytes, particularly basophils, can capture HIV-1 and mediate viral trans-infection of CD4(+) T cells. The expression of a variety of HIV-1 attachment factors, such as the C-type lectins, etc., facilitates viral capture and transfer. Intriguingly, the frequency of basophils in patients with different levels of CD4(+) T counts remains fairly stable during the course of disease. Our results provide novel insights into the roles of granulocytes, particularly basophils, in HIV-1 dissemination. We suggest that strategies designed to prevent basophil-mediated viral capture and transfer may be a new direction for the development of anti-HIV therapy.