Abstract Natural killer (NK) cells in the mouse liver sinusoid are divided into CD49a+DX5(CD49b)- and CD49a-DX5+, whereas those in the blood are mostly CD49a-DX5+. We therefore investigated whether human NK cells are also separated into distinct subsets according to CD49a/b expression. Peripheral blood mononuclear cells (PBMC) and intrasinusoidal lymphocytes (ISL) from healthy human donors were used for phenotypic and functional characterization of NK cells. Primary human NK cells expressed CD49a and CD49b and could be split into four subsets: CD49a+CD49b-, CD49a-CD49b+, CD49a+CD49b+, and CD49a-CD49b-. Frequencies of human NK cell subsets, however, differed from mouse NK cell subsets, as majority of NK cells in human PBMC are CD49a-CD49b- or CD49a+CD49b-. NK cells in ISL expressed lower CD49b but higher CD49a than those in PBMC. In addition, CD49a+ subset showed higher affinity to collagen. CD49b+ NK cells expressed more activation marker CD69 and inhibitory receptor ILT2, indicating they are activated subsets. While degranulation capacity was not affected by CD49a/b expression, CD49a-b+ subset produced higher IFN-γ and CD49a+b+ subset expressed more TRAIL upon activation. In conclusion, human NK cells were divided into four subsets based on CD49a/b expression. It appeared that NK cells in the liver have higher capacity to bind to extracelluar matrix. Small amount of CD49b+ NK cell subsets with activated phenotype were found in the blood but healthy liver had few CD49b+ NK cells.