Abstract Invariant Natural Killer T (iNKT) cells are potent regulators of diverse immune responses. Our previous study indicated that lack of miRNAs following the deletion of Dicer, a miRNAs-processing enzyme, affect iNKT cell development and function. However, the roles of specific miRNA in iNKT cell development and function has not been reported. To identify which miRNAs are involved in NKT cell development, we first performed a genome-wide miRNA expression profiling during NKT cell development and found that miR-155 was highly expressed in early iNKT cell development, followed by a sharp decrease in late developmental stage in thymus. To test the role of miR-155 in iNKT cells, two miR155 mutant mouse models had been used. With a conventional miR-155 deficient mouse model, we did not find remarkable defects of iNKT cell development and function. However, overexpression of miR-155 in bone marrow or thymus results in iNKT cell developmental defect in the thymus without dramatic effects on other immune cells. Furthermore, miR-155 overexpressed iNKT cells display profound defects in their cytokine production. Bone marrow from miR-155 overexpressed mice poorly reconstituted iNKT cells, suggesting a cell intrinsic effect. Finally, we have identified Itk (Inducible T cell Kinase) as a direct target of miR-155 in the regulation of iNKT cell development and function. Together, our data suggest that dynamic regulation of miR-155 expression is required for iNKT cell development and function.