Human Cytomegalovirus (HCMV) infection of monocytes results in the production of inflammatory cytokine interleukin-1β (IL-1β). The objective of the study was to explore the mechanism behind the activation of inflammation. HCMV infection activated the NLRP3 inflammasome and enhanced the secretion of IL-1β in THP-1 cells. Besides, HCMV infection caused mitochondrial dysfunction, including excessive ROS production, decreased mitochondrial membrane potential, and increased mitochondrial fusion. Importantly, the infection reduced the expression of TFAM protein and the release of mitochondrial DNA into the cytoplasm, which may contribute to the activation of NLRP3 inflammasome. Collectively, our finding suggests that HCMV infection may activate NLRP3 inflammasome by releasing mtDNA into the cytosol in human THP-1 cells and provides novel insights into the innate immune response against HCMV infection.