TSC2, or tuberin, is the product of the tuberous sclerosis tumor suppressor gene TSC2 and acts downstream of the phosphatidylinositol 3-kinase-Akt signaling pathway to negatively regulate cellular growth. One mechanism underlying its function is to assemble into a heterodimer with the TSC1 gene product TSC1, or hamartin, resulting in a reduction in phosphorylation, and hence activation, of the ribosomal subunit S6 kinase (S6K). We identified a novel interaction between TSC2 and 14-3-3beta. We found that 14-3-3beta does not interfere with TSC1-TSC2 binding and can form a ternary complex with these two proteins. Association between 14-3-3beta and TSC2 requires phosphorylation of TSC2 at a unique residue that is not a known Akt phosphorylation site. The overexpression of 14-3-3beta compromises the ability of the TSC1-TSC2 complex to reduce S6K phosphorylation. The antagonistic activity of 14-3-3beta toward TSC is dependent on the 14-3-3beta-TSC2 interaction, since a mutant of TSC2 that is not recognized by 14-3-3beta is refractory to 14-3-3beta. We suggest that 14-3-3 proteins interact with the TSC1-TSC2 complex and negatively regulate the function of the TSC proteins.