Many viruses have evolved mechanisms to alter mitochondrial function. The hepatitis C virus (HCV) produces a viral core protein that targets to mitochondria and increases Ca²⁺-dependent ROS production. The aim of this study was to determine whether core’s effects are mediated by changes in mitochondrial Ca²⁺ uptake. Core expression caused enhanced mitochondrial Ca²⁺ uptake in response to ER Ca²⁺ release induced by thapsigargin or ATP. It also increased mitochondrial superoxide production and mitochondrial permeability transition (MPT). Incubating mouse liver mitochondria with an HCV core (100 ng/mg) in vitro increased Ca²⁺ entry rate by 2-fold. Entry was entirely inhibited by the mitochondrial Ca²⁺ uniporter inhibitor, Ru-360, but not influenced by an Na⁺/Ca²⁺ exchanger inhibitor or ROS scavengers. These results indicate that core directly increases mitochondrial Ca²⁺ uptake via a primary effect on the uniporter. This enhanced the ability of mitochondria to sequester Ca²⁺ in response to ER Ca²⁺ release, and increased mitochondrial ROS production and MPT. Thus, the mitochondrial Ca²⁺ uniporter is a newly identified target for viral modification of cell function.--Li, Y., Boehning, D. F., Qian, T., Popov, V. L., Weinman. S. A. Hepatitis C virus core protein increases mitochondrial ROS production by stimulation of Ca²⁺ uniporter activity.