Hepatitis C virus (HCV) infection is one of the major factors to trigger a sustained hepatic inflammatory response and hence hepatocellular carcinoma (HCC), but direct-acting-antiviral (DAAs) was not efficient to suppress HCC development. Heat shock protein 90 kDa (HSP90) is highly abundant in different types of cancers, and especially controls protein translation, endoplasmic reticulum stress, and viral replication. In this study we investigated the correlation between the expression levels of HSP90 isoforms and inflammatory response marker NLRP3 in different types of HCC patients as well as the effect of a natural product celastrol in suppression of HCV translation and associated inflammatory response in vivo. We identified that the expression level of HSP90β isoform was correlated with that of NLRP3 in the liver tissues of HCV positive HCC patients (R² = 0.3867, P < 0.0101), but not in hepatitis B virus-associated HCC or cirrhosis patients. We demonstrated that celastrol (3, 10, 30 μM) dose-dependently suppressed the ATPase activity of both HSP90α and HSP90β, while its anti-HCV activity was dependent on the Ala47 residue in the ATPase pocket of HSP90β. Celastrol (200 nM) halted HCV internal ribosomal entry site (IRES)-mediated translation at the initial step by disrupting the association between HSP90β and 4EBP1. The inhibitory activity of celastrol on HCV RNA-dependent RNA polymerase (RdRp)-triggered inflammatory response also depended on the Ala47 residue of HSP90β. Intravenous injection of adenovirus expressing HCV NS5B (pAde-NS5B) in mice induced severe hepatic inflammatory response characterized by significantly increased infiltration of immune cells and hepatic expression level of Nlrp3, which was dose-dependently ameliorated by pretreatment with celastrol (0.2, 0.5 mg/kg, i.p.). This study reveals a fundamental role of HSP90β in governing HCV IRES-mediated translation as well as hepatic inflammation, and celastrol as a novel inhibitor of HCV translation and associated inflammation by specifically targeting HSP90β, which could be developed as a lead for the treatment of HSP90β positive HCV-associated HCC.