Invariant Vα14 natural killer T (Vα14i NKT) cells are a unique immunoregulatory T‐cell population that is restricted by CD1d. The glycolipid α‐galactosylceramide (α‐GalCer) is presented by CD1d and causes robust Vα14i NKT‐cell activation. Three days after injection of α‐GalCer, Vα14i NKT cells vigorously increase in number and then gradually decrease to normal levels. In the present study, we found that the re‐administration of α‐GalCer into mice primed 3 days earlier causes a marked increase in serum interleukin‐4 and interferon‐γ. Intracellular staining revealed that the only expanded Vα14i NKT cells are responsible for the enhanced cytokine production. The enhanced cytokine production was correlated with an increased number of Vα14i NKT cells after priming. Additionally, primed Vα14i NKT cells produced larger amounts of cytokine as compared with naive Vα14i NKT cells when cultured with α‐GalCer‐pulsed dendritic cells. Thus, we considered that a subset of expanded Vα14i NKT cells acquired a strong ability to produce cytokines. In contrast to mice primed 3 days earlier, cytokine production is markedly diminished in mice primed 7 days earlier. The expanded Vα14i NKT cells altered the surface phenotype (NK1.1^– CD69^–) and contained intracellular interferon‐γ. Additionally, we found that primed Vα14i NKT cells did not disappear or down‐regulate surface TCR expression when re‐injected with α‐GalCer as compared with naive Vα14i NKT cells. These results demonstrate that the function and surface phenotype of Vα14i NKT cells is dramatically altered after α‐GalCer priming.