Widespread applications of nanosized materials over the past decade have prompted investigations of desirable properties and potential hazards to humans and the environment. Titanium dioxide (TiO2) nanoparticles are one of the most widely used nanoparticles. To investigate the effect of biological functions induced by TiO2 nanoparticles (10 nm: TiO2 NPs) on human liver cell lines, normal liver cell line L02 and hepatoma cell line HepG2 were co-cultured with exogenous TiO2 NPs. Cell growth and proliferation, cell cycle, and the apoptosis rate were analyzed. The effects of TiO2 NPs on the expression levels of apoptosis-associated protein caspase-3 and the membrane channel protein ENaC and caspase-3/7 activity were determined. Moreover, the influence of TiO2 NPs on the expression levels of the mitochondria-related proteins SIRT3, VDAC1, and ACSS1, the mitochondrial membrane potential and the ADP/ATP ratio were also examined. Our results revealed that TiO2 NPs inhibited the growth and proliferation of HepG2 cells, suppressed the S phase of cell cycling, and induced apoptosis of HepG2 cells. Following an increase in concentration, the inhibitory effect induced by TiO2 NPs on proliferation and cell cycle was more evident, and the apoptosis rate increased in a significant concentration-dependent manner, whereas there was no significant effect on the growth, proliferation, apoptosis, and cell cycle of L02 cells. In addition, the results of western blot showed that in HepG2 cells, TiO2 NPs upregulated the expressions of the apoptosis-related protein caspase-3 and the membrane channel protein ENaC in a concentration-dependent manner. However, in L02 cells, there was no significant difference in the expression levels of caspase-3 or ENaC. Furthermore, TiO2 NPs induced depolarization of the mitochondrial membrane, upregulated the expression levels of the mitochondria-related proteins SIRT3 and VDAC1, and downregulated the expression level of the key respiratory chain protein ACSS1 in HepG2 cells. However, in L02 cells, the expressions of SIRT3, VDAC1, and ACSS1 exhibited no clear change. The apoptosis of HepG2 cells induced by TiO2 NPs may be achieved by regulating intracellular osmotic pressure; moreover, upregulating the expression of the channel protein ENaC or the mitochondrial porin VDAC1 and depolarizing the mitochondrial membrane of HepG2 cells resulted in the loss of Cyt-c and ATP and further activated caspase-3. To further confirm the above results, a nude mouse xenograft model was employed. After a certain period of treatment with TiO2 NPs, the nude mice were sacrificed, tumors were removed, and the expression of related proteins was detected. Immunohistochemistry and western blot results showed that the expressions of the proteins VDAC1 and SIRT3 were clearly upregulated in tissues treated to TiO2 NPs, whereas the expression of ACSS1 was downregulated. The results were consistent with the above in vitro results. All the above results confirmed that TiO2 NPs can act as a safe antitumor agent.