l-arginine (l-Arg) plays a central role in several biologic systems including the regulation of T-cell function. l-Arg depletion by myeloid-derived suppressor cells producing arginase I is seen in patients with cancer inducing T-cell anergy. We studied how l-Arg starvation could regulate T-cell–cycle progression. Stimulated T cells cultured in the absence of l-Arg are arrested in the G₀-G₁phase of the cell cycle. This was associated with an inability of T cells to up-regulate cyclin D3 and cyclin-dependent kinase 4 (cdk4), but not cdk6, resulting in an impaired downstream signaling with a decreased phosphorylation of Rb protein and a low expression and binding of E2F1. Silencing of cyclin D3 reproduced the cell cycle arrest caused by l-Arg starvation. The regulation of cyclin D3 and cdk4 by l-Arg starvation occurs at transcriptional and posttranscriptional levels. Signaling through GCN2 kinase is triggered during amino acid starvation. Experiments demonstrated that T cells from GCN2 knock-out mice did not show a decreased proliferation and were able to up-regulate cyclin D3 when cultured in the absence of l-Arg. These results contribute to the understanding of a central mechanism by which cancer and other diseases characterized by high arginase I production may cause T-cell dysfunction.