Rotenone, a classical inhibitor of complex I in the mitochondrial electron transport chain, is known for its ability to induce the production of reactive oxygen species (ROS) by disrupting electron transport within mitochondria. Despite its well-established role in inducing apoptosis and inflammation, the precise biological mechanisms underlying the effects of rotenone remain elusive.  In this study, we investigated the relationship between the signaling pathways induced by rotenone and NLRP3 inflammasome activation. Our findings reveal that Rotenone upregulates the cGAS-STING pathway, leading to increased accumulation of STING downstream proteins in THP-1 cells. Treatment with cyclosporin A (CsA), a mitochondrial permeability transition pore inhibitor, effectively blocked Rotenone-induced cGAS-STING pathway activation and IL-1β release. Furthermore, we observed a decrease in cleaved-caspase-1 upon CsA treatment. Notably, STING inhibitor H-151, effectively blocked IL-1β release and NLRP3 accumulation. Additionally, we observed the migration of the STING downstream protein NF-κB from the cytosol to the nucleus, involving its crucial role in NLRP3 inflammasome priming signaling. In summary, our findings indicate that Rotenone induces NLRP3 inflammasome activation in THP-1 cells through the upregulation of the cGAS-STING pathway.