The tyrosine kinase, ζ-chain-associated protein of 70 kDa (ZAP70), is essential for T cell development and activation. However, it remains elusive whether a direct redox regulation affects ZAP70 activity upon TCR stimulation. Here, we showed that deficiency of non-selenocysteine containing phospholipid hydroperoxide glutathione peroxidase (NPGPx), a redox sensor, resulted in T cell hyperproliferation and elevated cytokine productions. Through proteomic approaches, ZAP70 is identified as the key interacting protein of NPGPx through disulfide bonding. Here we utilized the MS/MS spectra to identify the cysteine residues in ZAP70 for NPGPx-mediated regulation. Combined molecular approaches, our results elucidate a delicate redox mechanism that NPGPx serves as a modulator to curb ZAP70 functions in maintaining T cell homeostasis.