To accomplish fertilization in the oviductal ampulla, ejaculated sperm are required to migrate through the female reproductive tract. However, this fundamental process largely remains unknown. In this study, we focused on the role of oviductal smooth muscle (myosalpinx) contractions in the sperm migration. Administration of prifinium bromide, padrin, to mice effectively suppressed myosalpinx contractions, resulting in a decreased rate of fertilization in a dose-dependent manner, and an abrogation of high-speed back-and-forth/shuttling flows of oviductal fluids around the isthmus. Regardless of padrin administration, no shuttling flows were found near the ampulla. In the isthmus, sperm formed a tight assemblage that was synchronized with the shuttling flows. The sperm assemblage was gradually loosened and then completely abolished near the ampulla. No sperm assemblage was formed in the isthmus when padrin was administrated. These results suggest that myosalpinx contractions play important roles in the formation of sperm assemblage in the isthmus, and in the transport of the assemblage to the middle region of the oviduct. It is also suggested that the motility of sperm is essential for the migration of sperm from the middle oviductal region to the ampulla.