Human reticulocyte 15-lipoxygenase (15-LOX) plays an important role in inflammation resolution and is also involved in many cancer-related processes. Both an activator and an inhibitor will serve as research tools for understanding the biological functions of 15-LOX and provide opportunities for drug discovery. In a previous study, both allosteric activators and inhibitors of 15-LOX were discovered through a virtual screening based computational approach. However, why molecules binding to the same site causes different effects remains to be disclosed. In the present study, we used previously reported activator and inhibitor molecules as probes to elucidate the mechanism of allosteric regulation of 15-LOX. We measured the influences of the allosteric activator and inhibitor on the enzymatic reaction rate and found that the activator increases 15-LOX activity by preventing substrate inhibition instead of increasing the turnover number. The inhibitor can also prevent substrate inhibition but decreases the turnover number at the same time, resulting in inhibition. Molecular dynamics simulations were conducted to help explain the underlying mechanism of allostery. Both the activator and inhibitor were demonstrated to be able to prevent 15-LOX from transforming into potentially inactive conformations. Compared to the activator, the inhibitor molecule restrains the motions of residues around the substrate binding site and reduces the flexibility of 15-LOX. These results explained the different effects between the activator and the inhibitor and shed light on how to effectively design novel activator molecules.