SWEETs and their prokaryotic counterparts SemiSWEETs were recently classified as transporters that translocate sugar across cellular membranes. SemiSWEETs are commonly used as a model system to infer biological properties of SWEETs; however, this presumes that the homologues are comparable to begin with. We evaluate this presumption by comparing their protein dynamics and substrate transport mechanism using 532 μs of simulation data in conjunction with Markov state models (MSMs). MSM weighted conformational landscape plots reveal significant differences between SWEETs and SemiSWEETs despite having similar structural topology. The presence of glucose reduces the free energy barrier between the functionally important intermediate states to enhance the transport process, while the substrate has no effect on SemiSWEET. The glucose adopts more rotational degrees of freedom in SWEET, while its conformation is restricted for SemiSWEET. Our study provides biological insights on the unexplored novelty of difference in the functional mechanism of two close homologous proteins.