Natural killer (NK) cells are believed to play a role in human immunodeficiency virus 1 (HIV-1) disease progression, and NK cell levels are reduced in individuals with chronic HIV-1 infection. In the present study, we compared the frequency and phenotype of peripheral blood CD3-CD56+ NK cells in HIV-1 infected patients in China who were infected through different routes of transmission, including heterosexual and homosexual sexual contact, and blood transmission through injection drug use or importation of blood or blood products. The results showed significantly reduced numbers of CD3-CD56+ NK cells with no association with route of transmission. The expression of CD16 on CD3-CD56+ NK cells in HIV-1 infected patients was similar to that in healthy controls. Among the examined receptor (KIR3DL1, NKp80, NKp44, CD244, NKG2D, and NTBA) expressions, only KIR3DL1 and NKp80 expressions on CD3-CD56+ NK cells were suppressed in HIV-1-infected patients compared to healthy controls, and no significant difference was observed between patients upon comparison of different routes of transmission. A subset of CD3(dim)/CD56+ cells was dramatically increased in HIV-1-infected patients. This study suggests that changes in NK cell count and receptors are not related to the route of HIV-1 transmission. A new subset of CD3(dim)/CD56+ cells emerged only in HIV-1-infected patients, and may play a role in limiting viral spread, eliminating infected cells, and slowing the progression from HIV-1 infection to AIDS.