Human invariant Vα24+ NKT cells are a relatively new subpopulation of lymphocytes. It has been reported that Vα24 NKT cells are significantly involved in some human diseases. We have evaluated the number and function of Vα24 NKT cells in both healthy volunteers and cancer patients. In this study we found that Vα24 NKT cells in unfractionated PBMCs obtained from cancer patients did not respond efficiently to α-galactosylceramide (α-GalCer) in vitro. Thus, their proportion after stimulation with α-GalCer was smaller than that found in healthy volunteers. However, the cancer patients’ Vα24 NKT cells retained cytotoxic activity against malignant target cells, and they could efficiently proliferate to α-GalCer when fractionated by sorting. Furthermore, we found that addition of G-CSF to the culture could restore the low proliferative response of Vα24 NKT cells from cancer patients. These results suggest that some functions of NKT cells in cancer patients are impaired, and this observation carries significant implications for immunotherapy-based cancer treatments.