CatSper channel, a cation channel expressed specifically in testis, is essential for fertilization in mice. It is believed that CatSper channel is formed by four α subunits, CatSper1, CatSper2, CatSper3 and CatSper4, as a hetero tetramer. Each subunit has six trans-membrane segments (S1-S6). Some features of the primary structure, such as positively charged amino acids in the fourth trans-membrane segment (S4) and the acidic residue in selectivity filter in the putative pore forming region of S5-S6, suggest that CatSper is a calcium-permeable voltage-dependent channel. However, detailed molecular characteristics of CatSper are still unclear since functional analysis using heterologous expression systems has been unsuccessful. In this study, we report that the region truncated just downstream of S4 of one isoform of ascidian CatSper orthologues, Ci-CatSper3, corresponding to the voltage-sensor domain of other voltage-gated ion channels, has Ca2+ permeability. Xenopus oocytes expressing the voltage sensor domain of Ci-CatSper3 (CiCS3 VSD) showed substantial ionic currents upon hyperpolarization of membrane potential. Calcium photometry using a calcium indicator revealed that Ca2+ permeable pathway was formed by CiCS3 VSD. Furthermore, by Ca2+ influx assay in liposome reconstitution system, it was demonstrated that CiCS3 VSD itself has Ca2+ permeability. This is, to our knowledge, the first study which reports calcium permeation through a voltage sensor domain.