Spirura genus Blanchard, 1849 comprise of nematode parasites that infect primate and marsupial species. Although several taxonomical studies have shown that the infection by this species occurs primarily in the esophagus of primates, evidence for the occurrence of these parasites in other hosts (marsupials, rodents and bats) has become the subject of investigation by several groups. In this work, we describe the presence of Spirura guianensis Ortlepp, 1924 in the marsupial Gracilinanus agilis (Marsupialia: Didelphidae) found in the Pantanal of Mato Grosso do Sul state of Brazil. Structural characteristics of this nematode were identified using light microscopy (bright field and fluorescence stereomicroscopy) and scanning electron microscopy (SEM) approaches. Details of the surface topography such as cephalic projections, ventral boss, details of the caudal papillae and cuticular ornamentations were shown, providing taxonomic characteristics that may help in the establishment of diagnostic protocols. In addition, the presence of this species in a new host and new geographical area of Brazil provide grounds for a revision on the distribution of S. guianensis in South America.