Myelin-derived Nogo-A protein limits axonal growth after CNS injury. One domain binds to the Nogo-66 receptor to inhibit axonal outgrowth, whereas a second domain, Amino-Nogo, inhibits axonal outgrowth and cell adhesion through unknown mechanisms. Here, we show that Amino-Nogo inhibition depends strictly on the composition of the extracellular matrix, suggesting that Amino-Nogo inhibits the function of certain integrins. Amino-Nogo inhibition can be partially overcome by antibodies that activate integrin beta1 or by the addition of Mn2+, an integrin activator. Furthermore, Amino-Nogo reduces focal adhesion kinase activation by fibronectin. Analysis of various cell lines reveals that alpha(v)beta3, alpha5, and alpha4 integrins are sensitive to Amino-Nogo, but alpha6 integrin is not. Both alpha(v) and alpha5 integrins have widespread expression in adult brain and are found in axonal growth cones. Thus, inhibition of integrin signaling by Amino-Nogo contributes to the failure of CNS axon regeneration.