We explored lithium ion transport phenomena in a cation-deficient quadruple perovskite LiCuTa3O9 based on ab initio calculations and experiments. Here, we investigated the stability of cation-ordered/disordered phases by employing a Li–Cu–vacancy ternary lattice model and canonical Monte Carlo simulations. Our studies predict the formation of interconnected chains of lithium and vacancies, extending along all crystallographic axes, in the most stable cation-ordered phase. We have investigated and discussed the mechanism of lithium diffusion through this system by calculating the activation energy associated with the localized nearest neighbor lithium hoppings between active sites. In our investigations, we measured lithium ion conductivity in the epitaxial thin films of LiCuTa3O9 fabricated on YAlO3 (110). The film, as investigated by reciprocal space mapping of X-ray diffraction, showed in-plane lattice anisotropy induced by the substrate. Notably, a small anisotropy in lithium transport was also observed.