Interleukin-1β (IL-1β) is a potent pro-inflammatory cytokine involved in the pathogenesis of HCV, but the sensors and underlying mechanisms that facilitate HCV-induced IL-1β proteolytic activation and secretion remains unclear. In this study, we have identified a signalling pathway leading to IL-1β activation and secretion in response to HCV infection. Previous studies have shown the induction and secretion of IL-1β through the inflammasome complex in macrophages/monocytes. Here, we report for the first time the induction and assembly of the NALP3-inflammasome complex in human hepatoma cells infected with HCV (JFH-1). We demonstrate that activation of IL-1β in HCV-infected cells involves the proteolytic processing of pro-caspase-1 into mature caspase-1 in a multiprotein inflammasome complex. Next, we demonstrate that HCV is sensed by NALP3 protein, which recruits the adaptor protein ASC for the assembly of the inflammasome complex. Using a small interfering RNA approach, we further show that components of the inflammasome complex are involved in the activation of IL-1β in HCV-infected cells. Our study also demonstrates the role of reactive oxygen species in HCV-induced IL-1β secretion. Collectively, these observations provide an insight into the mechanism of IL-1β processing and secretion, which is likely to provide novel strategies for targeting the viral or cellular determinants to arrest the progression of liver disease associated with chronic HCV infection.