Highly efficient catalytic oxidation toward cyclohexane with molecular oxygen (O₂) is still one of the most important reactions in current catalytic chemistry. Demonstrated here is how to effectively enhance the catalytic aerobic oxidation of cyclohexane over a mixed-valence {V₁₆} cluster-based metal–organic framework (NENU-MV-1) through rational size regulation. The average particle size of NENU-MV-1 was tuned notably from 25 μm to 500 nm by changing reaction temperature, concentration and molar ratio of metal source to ligand during solvothermal reactions. Remarkably, NENU-MV-1 with 500 nm particle size yields a higher conversion efficiency of cyclohexane (24.6%) and selectivity of the mixture of cyclohexane and cyclohexone (99%) than that of bulk crystalline NENU-MV-1. The kinetic study signified that catalysts with smaller particle size showed a faster catalytic rate, which is mainly attributed to the fact that catalysts with smaller particle size are able to expose more active sites and then more fully contact with substrate molecules. In addition, the mixed-valence {V₁₆} cluster-based oxidation catalysts exhibited higher activity than the single-valence vanadium cluster-based oxidation catalysts and V^IV and V^V-based physical mixture. An in-depth investigation of the reason for the high reactivity of NENU-MV-1 indicated that there may be a synergistic catalytic effect of V^IV–V^V centers.