Titanium dioxide nanoparticles (nano-TiO), as a common nanomaterial, are widely used in water purification, paint, skincare and sunscreens. Its safety has always been a concern. Prior studies have shown that ultraviolet A (UVA) can exacerbate the toxicity of nano-TiO, including inducing cell apoptosis, changing glycosylation levels, arresting cell cycle, inhibiting tumor cell and bacterial growth. However, whether the combination of UVA and nano-TiO cause cell necrosis and its mechanism are still rarely reported. In this study, we investigated the cytotoxicity and phototoxicity of mixture crystalline nano-TiO (25% rutile and 75% anatase, 21 nm) under UVA irradiation in HeLa cells. Our results showed that the abnormal membrane integrity and the ultrastructure of HeLa cells, together with the decreased viability induced by nano-TiO under UVA irradiation, were due to cell necrosis rather than caspase-dependent apoptosis. Furthermore, nano-TiO and UVA generated the reactive oxygen species (ROS) and caused the mitochondrial permeability transition pore (mPTP) of HeLa cells to abnormally open. Cell viability was significantly increased after adding vitamin C (VC) or cyclosporin A (CsA) individually to inhibit ROS and mPTP. Clearance of ROS could not only impede the opening of mPTP but also reduce the rate of cell necrosis. The results suggest the possible mechanism of HeLa cell necrosis caused by nano-TiO under UVA irradiation through the ROS-mPTP pathway.