Proliferative vitreo retinopathy (PVR) is one of the ocular complications, marked by the enhanced proliferation of various cells including retinal pigment epithelial cells (RPE). The aim of the present study is to analyze the effect of gold nanoparticles (Au-NP) on vascular endothelial growth factor (VEGF) and interleukin-1 beta (IL-1β)-induced cell spreading, migration and proliferation in RPE cells. Au-NP (300 nM) significantly blocked the VEGF-and IL-1β-induced cell spreading, migration and proliferation in bovine RPE cells (BRPEs). To elucidate the signaling mechanism of VEGF- and IL-1β-induced cell proliferation, BRPEs were treated with PP2, a Src inhibitor. Further, to clarify the possible involvement of the Src pathway on the inhibitory effect of Au-NPs, transient transfection assay was performed using dominant negative (DN) and constitutively active (CA) mutant plasmid of Src kinase. The results showed that VEGF and IL-1β exert their proliferative effects through the activation of Src kinase whereas CA Src rescued the inhibitory effect of Au-NP in presence or absence of VEGF and IL-1β in BRPEs. Further, an in vitro kinase assay was performed to identify the status of Src phosphorylation at Y419. We found that VEGF and IL-1β increased Src phosphorylation in BRPEs and Au-NP blocked the VEGF- and IL-1β-induced Src phosphorylation at Y419. Taken together, our result suggests that Au-NP could effectively inhibit the VEGF- and IL-1β-induced proliferation and migration by suppressing the Src kinase pathway in BRPEs and Au-NP might act as an effective therapeutic agent for the treatment of ocular diseases such as proliferative vitreo retinopathy.