A laboratory study was undertaken to explore the influential effects of mass transfer resistance on overall substrate removal in acetate-fed and glucose-fed sequential aerobic sludge blanket reactors. In both reactors, solids retention time decreased with increasing OLR [2–8 kg chemical oxygen demand (COD)/m3 d], resulting in increasing specific substrate utilization rates. The obtained kinetic parameters values (k/Ks ratio) indicated that the microbial reaction rate for acetate was higher than that for glucose. The simulated mass transfer parameter values (2, Bi, L, and ) and substrate concentration profiles in the granule indicated that the overall substrate removal in the acetate-fed and glucose-fed reactors are intra-granular diffusion controlled, and the influential effect of intra-granular mass transfer resistance in the glucose-fed reactor is relatively greater. The simulated results also disclosed that the optimal dp for acetate-fed and glucose-fed reactors should be no greater than 3.5 and 2.5 mm, respectively. The validated kinetic model and the obtained kinetic parameter values can be appropriately used to simulate treatment performance of the SASB reactors treating simple substrates.