Polymerized type I collagen suppresses fibroblast proliferation. Previous studies have implicated inhibition of fibroblast proliferation with polymerized collagen-mediated suppression of S6K1, but the molecular mechanism of the critical negative feedback loop has not yet been fully elucidated. Here, we demonstrate that polymerized collagen suppresses G(1)/S phase transition and fibroblast proliferation by a novel mechanism involving the formation of a beta1 integrin-protein phosphatase 2A (PP2A)-tuberous sclerosis complex 2 (TSC2) complex that represses S6K1 activity. In response to fibroblast interaction with polymerized collagen, beta1 integrin forms a complex with PP2A that targets TSC2 as a substrate. PP2A represses the level of TSC2 phosphorylation and maintains TSC2 in an activated state. Activated TSC2 negatively regulates the downstream kinase S6K1 and inhibits G(1)/S transit. Knockdown of TSC2 enables fibroblasts to overcome the anti-proliferative properties of polymerized collagen. Furthermore, we show that this reduction in TSC2 and S6K1 phosphorylation occurs largely independent of Akt. Although S6K1 activity was markedly suppressed by polymerized collagen, we found that minimal changes in Akt activity occurred. We demonstrate that up-regulation of Akt by overexpression of constitutively active phosphatidylinositol 3-kinase p110 subunit had minor effects on TSC2 and S6K1 phosphorylation. These findings demonstrate that polymerized collagen represses fibroblast proliferation by a mechanism involving the formation of a beta1 integrin-PP2A-TSC2 complex that negatively regulates S6K1 and inhibits G(1)/S phase transition.