A description is provided for Gracilioxyuris agilisis n. g., n. sp. found in the cecum of Gracilinanus agilis Burmeister, 1854 (Gardner, 2005) (Didelphidae) in Brazilian Pantanal. The new species can be distinguished from Didelphoxyuris thylamisis by a dome-shaped anterior end, a weakly developed cephalic vesicle, males with no postcloacal ornamentation, and operculate eggs with 3 longitudinal ridges. Gracilioxyuris n. g. is characterized by a small and apical oral opening and double-crested lateral alae. Males possess an area rugosa as a ventral, keellike elevation with transverse striations, 4 pairs of genital papillae, the first and second pairs of which are adanal, and a third minute pair just posterior to cloacal aperture; the last pair are at the caudal end with no tail tip. Females possess a thick muscular vagina, a didelphic genital tract, and operculated eggs with ridges.