Natural killer (NK) cells play important roles in adoptive cellular immunotherapy against certain human cancers. This study aims to establish a new human NK cell line and to study its role for adoptive cancer immunotherapy. Peripheral blood samples were collected from 54 patients to establish the NK cell line. A new human NK cell line, termed as NKG, was established from a Chinese male patient with rapidly progressive non-Hodgkin's lymphoma. NKG cells showed LGL morphology and were phenotypically identified as CD56(bright) NK cell with CD16(-), CD27(-), CD3(-), αβTCR(-), γδTCR(-), CD4(-), CD8(-), CD19(-), CD161(-), CD45(+), CXCR4(+), CCR7(+), CXCR1(-), and CX3CR1(-). NKG cells showed high expression of adhesive molecules (CD2, CD58, CD11a, CD54, CD11b, CD11c), an array of activating receptors (NKp30, NKp44, NKp46, NKG2D, NKG2C), and cytolysis-related receptors and molecules (TRAIL, FasL, granzyme B, perforin, IFN-γ). The cytotoxicity of NKG cells against tumor cells was higher than that of the established NK cell lines NK-92, NKL, and YT. NKG cell cytotoxicity depended on the presence of NKG2D and NKp30. When irradiated with 8 Gy, NKG cells were still with high cytotoxicity and activity in vitro and with safety in vivo, but without proliferation. Further, the irradiated NKG cells exhibited strong cytotoxicity against human primary ovarian cancer cells in vitro, and against human ovarian cancer in a mouse xenograft model. The adoptive transfer of NKG cells significantly inhibited the ovarian tumor growth, decreased the mortality rate and prolonged the survival, even in cases of advanced diseases. A number of NKG cells were detected in the ovarian tumor tissues during cell therapy. In use of the new human NK cell line, NKG would a promising cellular candidate for adoptive immunotherapy of human cancer.