During development, dopaminergic neurons born in the substantia nigra extend their axons toward the striatum. However, the mechanisms by which the dopaminergic axons extend the striatum to innervate their targets remain unclear. We previously showed that paired-cultivation of mesencephalic cells containing dopaminergic neurons with striatal cells leads to the extension of dopaminergic neurites from the mesencephalic cell region to the striatal cell region. The present study shows that dopaminergic neurites extended along striatal neurons in the paired-cultures of mesencephalic cells with striatal cells. The extension of dopaminergic neurites was suppressed by the pharmacological inhibition of integrin α5β1. Using lentiviral vectors, short hairpin RNA (shRNA)-mediated knockdown of integrin α5 in dopaminergic neurons suppressed the neurite outgrowth to the striatal cell region. In contrast, the knockdown of integrin α5 in non-dopaminergic mesencephalic and striatal cells had no effect. Furthermore, overexpression of integrin α5 in dopaminergic neurons differentiated from embryonic stem cells enhanced their neurite outgrowth on striatal cells. These results indicate that integrin α5β1 expression on dopaminergic neurons plays an important role in the dopaminergic neurite outgrowth on striatal neurons.