Heterakis spumosa is a nematode of invasive rodents, mainly affiliated with Rattus spp. of Asian origin. Despite the ecological importance and cosmopolitan distribution, little information is available on the genetic characteristics and infectivity to experimental animals of this roundworm. Heterakis isolates obtained from naturally infected brown rats caught in 2007 in the city of Sagamihara, east central Honshu, Japan, and maintained by laboratory passages were subjected to mitochondrial sequence analysis and experimental infection in mice. Sequencing of the cox1 gene revealed that nucleotides of H. spumosa and previously examined Heterakis isolonche isolates from gallinaceous birds in Japan differed by 11.2-12.2% that conforms to the range expected for interspecific differences. The two H. spumosa isolates differed by a single 138T/C non-synonymous substitution in the 393-bp mt sequence. In a dendrogram, the H. spumosa samples formed a subcluster with members of the nematode superfamily Heterakoidea, H. isolonche and Ascaridia galli. In an experimental infection study, ICR, AKR, B10.BR and C57BL/6 mice strains were inoculated with 200 H. spumosa eggs/head and necropsied at 14 and 90 days post-inoculation (DPI) when the number of worms was recorded. Eggs were initially detected in faeces from 32-35 DPI in ICR, AKR and B10.BR mice and the highest mean number of eggs per gram of faeces (EPG) was 4,800 at 38 DPI, 2,200 at 58 DPI and 800 at 44 and 72 DPI in ICR, AKR and B10.BR mice, respectively. No eggs were observed in faeces of the C57BL/6 mouse strain during the experiment. A similar number of juvenile worms were isolated from all mouse strains at 14 DPI, whereas no adult worms were detected in C57BL/6 mice at 90 DPI.