Previous studies examined various immune evasion strategies of human cytomegalovirus (HCMV) to understand HCMV pathogenesis. Although the mechanism that underlies immunocyte destruction near HCMV-infected lesions has yet to be established, we show that substances produced by HCMV-infected cells induce death in several types of immunocytes, but not in fibroblasts or astrocytomas. These substances contain HCMV proteins and were termed HCMV-associated insoluble substance (HCMVAIS). We characterized this mechanism of cell death induced by HCMVAIS to understand the death of immunocytes near HCMV-infected lesions. HCMVAIS triggered the production of intracellular nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived reactive oxygen species (ROS), resulting in cell death, and the effect was reversed following treatment with ROS inhibitors. Cell death was not induced in splenocytes from NOX-2 knockout mice. We hypothesized that DNA damage induced by oxidative stress could initiate poly ADP-ribose polymerase-1 (PARP-1)-mediated cell death, or parthanatos. We report here HCMVAIS-induced cell death accompanied by PARP-1 activation in a caspase-independent manner, the nuclear translocation of apoptosis-inducing factor (AIF), and DNA fragmentation, which are typical features of parthanatos. Treatment with an AIF inhibitor decreased the rate of HCMVAIS-induced cell death. This event was confirmed by hematoxylin and eosin (H&E) staining; cell death in most HCMV-positive foci was TUNEL-positive, cleaved caspase 3-negative, and CD45-positive in serial section samples of a large intestine with HCMV infection. Taken together, these data suggest that HCMV inhibits local immune responses via direct killing of immunocytes in the vicinity of HCMV-infected cells through ROS-induced parthanatos by HCMVAIS.