Silver nanoparticles (AgNPs) have been reported to penetrate the central nervous system (CNS) and induce neurotoxicity. However, there is a paucity of understanding of the toxicity of AgNPs and their effect on the blood-brain barrier (BBB) including the underlying molecular mechanism(s) of action. Such information is important for the formulation of new strategies for delivery of biological therapeutics to central nervous system (CNS) targets. Using an in vitro BBB model and mass spectrometry-based proteomics, we investigated alterations in the proteomes of brain endothelial cells and astrocytes at different time points after AgNPs exposure (24 and 48 h). Our data showed that several proteins involved in neurodisorders and neurodegeneration were significantly upregulated in endothelial cells (e.g. 7-dehydrocholesterol reductase, zinc transporters 1 and 6), while proteins responsible for maintaining brain homeostasis were significantly downregulated (e.g anti-oxidative proteins glutathione peroxidase 1 and glutathione peroxidase 4). Many inflammatory pathways were significantly upregulated at 24 h post-AgNPs exposure (C9 pathway), while at 48 h proteins involved in BBB damage and anti-inflammatory responses were upregulated (quinoneoxidoreductase1 and glutamate cysteine ligase catalytic subunit) suggesting that by the later time point, cellular protection pathways had been activated to rescue the cells from AgNPs-induced toxicity. Our study suggests that in the initial stage of exposure, AgNPs exerted direct cellular stress on the endothelial cells by triggering a pro-inflammatory cascade. This study provides detailed insight into the toxic potency of AgNPs on in vitro BBB model and adds to the understanding of the adaptive role of BBB with regards to AgNPs-mediated toxicity.