Chikungunya virus (CHIKV), a positive-stranded RNA virus, can cause neurological complications by infecting the major parenchymal cells of the brain such as neurons and astrocytes. A proteomic analysis of CHIKV-infected human astrocytic cell line U-87 MG revealed tight functional associations among the modulated proteins. The predominant cellular pathways involved were of transcription–translation machinery, cytoskeletol reorganization, apoptosis, ubiquitination, and metabolism. In the proteome, we could also identify a few proteins that are reported to be involved in host–virus interactions. One such protein, Nucleophosmin (NPM1)/B23, a nucleolar protein, showed enhanced cytoplasmic aggregation in CHIKV-infected cells. NPM1 aggregation was predominantly localized in areas wherein CHIKV antigen could be detected. Furthermore, we observed that inhibition of this aggregation using a specific NPM1 oligomerization inhibitor, NSC348884, caused a significant dose-dependent enhancement in virus replication. There was a marked increase in the amount of intracellular viral RNA, and ∼105-fold increase in progeny virions in infected cells. Our proteomic analysis provides a comprehensive spectrum of host proteins modulated in response to CHIKV infection in astrocytic cells. Our results also show that NPM1/B23, a multifunctional chaperone, plays a critical role in restricting CHIKV replication and is a possible target for antiviral strategies.