The tyrosine kinase Zap-70 is a key regulator of T cell receptor (TCR) signaling downstream of antigen presentation, with coordinated regulation of Zap-70 kinase activity critical for proper T cell proliferation, differentiation, and effector function during an immune response. Zap-70 is cytosolic in unstimulated T cells, but is rapidly recruited to the TCR complex following receptor stimulation. Its activity is regulated both by binding to subunits of the TCR and by phosphorylation on multiple tyrosine residues. Zap-70 also has been reported to be ubiquitinated following TCR stimulation. Herein, we confirm the ubiquitination of Zap-70 in T cell lines and in primary human and mouse T cells, and report the identification of nine novel Zap-70 ubiquitination sites. Three sites, including Lys-193, Lys-217, and Lys-376, displayed greater than 20-fold increase in modification levels following TCR stimulation. Abrogation of Lys-217 ubiquitination results in increased kinase activation, enhanced activation of downstream signaling pathways, and elevated IL-2 production following TCR stimulation. These data suggest that Zap-70 ubiquitination contributes to the regulation of Zap-70 signaling following TCR stimulation.