Human cytomegalovirus (HCMV) infection of monocytes results in the production of inflammatory cytokine through inflammasome. However, the mechanism of NLR family pyrin domain containing 3 (NLRP3) inflammasome activation in HCMV infection remains unclear. In this study, HCMV infection promoted the increase of mitochondrial fusion and caused mitochondrial dysfunction in THP‐1 cells, including excessive reactive oxygen species production and decreased mitochondrial membrane potential (Δψm). Meanwhile, the expression of mitochondrial DNA (mtDNA)‐binding protein TFAM (transcription factor A, mitochondrial) was decreased and mtDNA content in the cytoplasm was increased. Knockdown of TFAM caused an increase in mtDNA copy number in the cytoplasm and resulted in elevated NLRP3 expression, active caspase‐1, and mature IL‐1β. After a 3 h treatment with MCC950, an NLRP3 inhibitor, the increase of cleaved caspase‐1 and mature IL‐1β were suppressed. Besides, overexpression of TFAM inhibited the expression of NLRP3, cleaved caspase‐1, and mature IL‐1β. In addition, knockdown of NLRP3 inhibited the IL‐1β process after HCMV infection. mtDNA‐deficient cells showed a limited ability to produce NLRP3 and process IL‐1β after HCMV infection. In conclusion, HCMV infection of THP‐1 cells resulted in decreased mitochondrial TFAM protein expression and increased mtDNA release into the cytoplasm, which eventually led to the activation of NLRP3 inflammasome.