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Spermatozoa sense and respond to their environmental signals to ensure fertilization success. Reception and transduction of signals are reflected rapidly in sperm flagellar waveforms and swimming behavior. In the ascidian <i>Ciona intestinalis</i> (type A; also called <i>C. robusta</i>), an egg-derived sulfated steroid called SAAF (sperm activating and attracting factor), induces both sperm motility activation and chemotaxis. Two types of CNG (cyclic nucleotide-gated) channels, Ci-tetra KCNG (tetrameric, cyclic nucleotide-gated, K⁺-selective) and Ci-HCN (hyperpolarization-activated and cyclic nucleotide-gated), are highly expressed in <i>Ciona</i> testis from the comprehensive gene expression analysis. To elucidate the sperm signaling pathway to regulate flagellar motility, we focus on the role of CNG channels. In this study, the immunochemical analysis revealed that both CNG channels are expressed in <i>Ciona</i> sperm and localized to sperm flagella. Sperm motility analysis and Ca²⁺ imaging during chemotaxis showed that CNG channel inhibition affected the changes in flagellar waveforms and Ca²⁺ efflux needed for the chemotactic turn. These results suggest that CNG channels in <i>Ciona</i> sperm play a vital role in regulating sperm motility and intracellular Ca²⁺ regulation during chemotaxis.