Urochordates are marine animals, and spermatozoa of many urochordates show chemotactic behavior toward conspecific eggs during fertilization. Sperm chemotaxis of the ascidian Ciona intestinalis has been particularly well investigated. The sperm-activating and sperm-attracting factor (SAAF) of the phlebobranchian ascidians are secreted from the egg cells, and identified as polyhydroxylsterolsulfates. The molecular structure of SAAFs differs in different species, and the differences may cause species-specific chemotactic responses. Ascidian spermatozoa appear to sense a SAAF concentration decrease, resulting in a transient increases in intracellular Ca2+ in the flagellum and quick changes in the swimming direction of the sperm. In this chapter, we will introduce the features and molecular mechanisms of sperm chemotaxis in urochordates, and particularly those in ascidians.