T cells survival, proliferation and anti-tumor response are tightly linked to their mitochondrial health. Complement C1q binding protein (C1QBP) promotes the mitochondrial fitness through regulation of mitochondrial metabolism and morphology. However, whether C1QBP regulates T cells survival, proliferation and anti-tumor immune function remains unclear. Our data demonstrated that C1QBP knocking down induced the accumulation of reactive oxygen species (ROS) and the loss of mitochondrial membrane potential to impair T cells mitochondrial fitness. At the same time, C1QBP insufficiency led to the less recruitment of the anti-apoptotic proteins including Bcl-2 and Bcl-XL to repress caspase-3 activation and PARP cleavage, which consequently accelerated T cells apoptotic process. On the other hand, C1QBP knocking-down rendered T cells with the relatively weaker proliferation due to the inhibition of AKT/mTOR signaling pathway. In order to investigate the exact role of C1QBP in anti-tumor response, C1QBP+/- and C1QBP+/+ mice were given the subcutaneous injection of murine MC38 cells, respectively. We found that C1QBP deficiency attenuated T cells tumor infiltration and aggravated these tumor-infiltrating T lymphocytes (TILs) exhaustion. Moreover, we further clarified the potential function of C1QBP in the chimeric antigen receptor (CAR)-T cells immunotherapy. Our data showed that C1QBP+/- CAR-T cells exhibited relatively weaker anti-tumor response than the corresponding C1QBP+/+ CAR-T cells. Given that C1QBP knocking down impairs T cells anti-apoptotic capacity, proliferation as well as anti-tumor immune function, development of the strategy to potentiation of T cells mitochondrial fitness through C1QBP would have a promise to optimize their efficacy of the related immunotherapy.