The ZSM-12 zeolite has attracted attention as the promising acid component of bifunctional catalysts for the n -alkane hydroisomerization because of its large size of micropore openings (0.57 nm × 0.61 nm) with 12-membered-ring and one-dimensional channels. However, the larger crystal size and stronger Brønsted acid strength of microsized ZSM-12 zeolite will lead to cracking of iso -olefin intermediates and decrease the iso -alkane yield. In this study, ZSM-12 zeolite samples partially and completely isomorphously substituted with gallium ([Ga,Al]Z12 and GaZ12) were in situ synthesized. The characteristic results indicate that isomorphous substitution by Ga can effectively reduce the crystal size to increase the mesoporosity and weaken the Brønsted acidity of the [Ga,Al]Z12 and GaZ12 samples. In addition, bifunctional catalysts with more appropriate metal-acid proximity for n -hexadecane hydroisomerization were prepared by mixing the 0.6Pd/A sample with 0.6 wt.% Pd loaded on γ-Al2O3 and the ZSM-12, [Ga,Al]Z12 and GaZ12 samples, respectively. The 0.3Pd/A-[Ga,Al]Z12 and 0.3Pd/A-GaZ12 catalysts both promote the maximum iso -hexadecane yield and distribution of multi-branched iso -hexadecane products due to their enhanced mesoporosity, reduced Brønsted acid strength, increased CPd/CH+ ratios and improved metal-acid balance. Especially for the 0.3Pd/A-GaZ12 catalyst, when the n -hexadecane conversion is 93.5%, the maximum iso -hexadecane yield reaches 80.6%, and the proportion of multi-branched iso -hexadecane products is 64.6%, which are both the highest among all investigated catalysts. Accordingly, Ga isomorphous substitution is an effective strategy to develop the efficient bifunctional catalysts for hydroisomerization.