Mammalian arachidonic acid lipoxygenases (ALOXs) have been implicated in the pathogenesis of inflammatory diseases, and its pro- and anti-inflammatory effects have been reported for different ALOX-isoforms. Human ALOX15B oxygenates arachidonic acid to its 15-hydroperoxy derivative, whereas the corresponding 8-hydroperoxide is formed by mouse Alox15b (Alox8). This functional difference impacts the biosynthetic capacity of the two enzymes for creating pro- and anti-inflammatory eicosanoids. To explore the functional consequences of the humanization of the reaction specificity of mouseAlox15bin vivo, we testedAlox15bknock-in mice that express the arachidonic acid 15-lipoxygenating Tyr603Asp and His604Val double mutant ofAlox15b, instead of the arachidonic acid 8-lipoxygenating wildtype enzyme, in two different animal inflammation models. In the dextran sodium sulfate-induced colitis model, femaleAlox15b-KI mice lost significantly more bodyweight during the acute phase of inflammation and recovered less rapidly during the resolution phase. Although we observed significant differences in the colonic levels of selected pro- and anti-inflammatory eicosanoids during the time-course of inflammation, there were no differences between the two genotypes at any time-point of the disease. In Freund’s complete adjuvant-induced paw edema model,Alox15b-KI mice were less susceptible than outbred wildtype controls, though we did not observe significant differences in pain perception (Hargreaves-test, von Frey-test) when the two genotypes were compared. our data indicate that humanization of the reaction specificity of mouseAlox15b(Alox8) sensitizes mice for dextran sodium sulfate-induced experimental colitis, but partly protects the animals in the complete Freund’s adjuvant-induced paw edema model.