NKT cells contribute to the modulation of immune responses and are believed to be important in the pathogenesis of autoimmune and infectious diseases, as well as cancer. Variations in the composite NKT cytokine response may determine individual disease susceptibility or severity. Due to low frequencies in peripheral blood, knowledge of the breadth of ex vivo human NKT cell functions has been limited. To bridge this gap, we studied highly purified NKT cells from PBMC of healthy donors and assessed the production of 27 effector functions using sensitive Elispot and multiplex bead assays. We found the ex vivo human NKT cell response is predominantly comprised of the chemokines MIP1-α, and MIP1-β as well as the Th1 cytokines IFN-γ and TNF-α. Although lower in magnitude, there was also significant production of IL-2, IL-4, and perforin after mitogen stimulation. Surprisingly, little/no IL-5, IL-6, IL-10, or IL-13 was detected, and no subjects' NKT cells produced IL-17. Comparison of the NKT functional profiles between age-matched male and female subjects revealed similar IL-4 responses, but higher frequencies of cells producing IFN-γ and MIP1-α, from males. There were no gender differences in the circulating NKT subset distribution. These findings implicate chemokines as a major mechanism by which NKT cells control responses in humans. In addition, the panoply of Th2 and Th17 cytokine secretion by NKT cells from healthy donors may not be as pronounced as previously believed. NKT cells may therefore contribute to the gender bias found in many diseases.