Although pericentromeric heterochromatin is essential for chromosome segregation, its role in humans remains controversial. Dissecting the function of HIV-1–encoded Vpr, we unraveled important properties of heterochromatin during chromosome segregation. In Vpr-expressing cells, hRad21, hSgo1, and hMis12, which are crucial for proper chromosome segregation, were displaced from the centromeres of mitotic chromosomes, resulting in premature chromatid separation (PCS). Interestingly, Vpr displaced heterochromatin protein 1-α (HP1-α) and HP1-γ from chromatin. RNA interference (RNAi) experiments revealed that down-regulation of HP1-α and/or HP1-γ induced PCS, concomitant with the displacement of hRad21. Notably, Vpr stimulated the acetylation of histone H3, whereas p300 RNAi attenuated the Vpr-induced displacement of HP1-α and PCS. Furthermore, Vpr bound to p300 that was present in insoluble regions of the nucleus, suggesting that Vpr aberrantly recruits the histone acetyltransferase activity of p300 to chromatin, displaces HP1-α, and causes chromatid cohesion defects. Our study reveals for the first time centromere cohesion impairment resulting from epigenetic disruption of higher-order structures of heterochromatin by a viral pathogen.