The guinea pig, a widely used experimental animal, has been used in myopia research in recent years. The structure of the extraocular muscles is important in research on eyeball movement, regulation of movement, binocular vision and surgical intervention. In this study, the anatomy and the structure of the extraocular muscles of guinea pigs were investigated. Five guinea pig eyes were dissected under a surgical microscope immediately after sacrifice, and an additional five were fixed in 10% formaldehyde solution and dissected under a surgical microscope 1 week after sacrifice. The guinea pig eye has seven extraocular muscles: two medial rectus muscles, one superior rectus muscle, one inferior rectus muscle, one superior oblique muscle and one inferior oblique muscle. The retractor bulbi muscle fibers surround the optic nerve longitudinally and insert circumferentially into the posterior pole of the eyeball. The lateral rectus was not found. Our results showed that there is a disparity between the structure of guinea pig extraocular muscles and that of humans.