The processes of activation, extravasation, and migration of immune cells to a site are early and essential steps in the induction of an acute inflammatory response. These events are part of the inflammatory cascade which involves multiple regulatory steps. Using a murine air-pouch model of inflammation with LPS as an inflammation inducer we demonstrate that isoenzymes of the neuraminidase family (NEU1, 3, and 4) play essential roles in this process acting as positive or negative regulators of leukocyte infiltration. Genetically knocked-out (KO) mice for different NEU genes (Neu1 KO, Neu3 KO, Neu4 KO, and Neu3/4 double KO mice) were induced with LPS, leukocytes at the site of inflammation were counted, and the inflamed tissue was analyzed using immunohistochemistry. Our data show that leukocyte recruitment was decreased in NEU1 and NEU3-deficient mice, while it was increased in NEU4-deficient animals. Consistent with these results, systemic levels of pro-inflammatory cytokines and those in pouch exudate were reduced in Neu1 and increased in Neu4 KO mice. We found that pharmacological inhibitors specific for NEU1, NEU3, and NEU4 isoforms also affected leukocyte recruitment. We conclude that NEU isoenzymes have distinct – and even opposing – effects on leukocyte recruitment, and therefore warrant further investigation to determine their mechanisms and importance as regulators of the inflammatory cascade.