Abstract Background Chemokine (C-C motif) receptor 6 (CCR6) is present in sperm and plays a significant role in sperm motility and chemotaxis acting in the reproductive tracts. However, the expression and functional significance of CCR6 in testis are still poorly understood, especially in the process of spermatogenesis. Methods and results CCR6 was expressed in spermatogenic cell lines and its expression was shown in an age-dependent upregulation manner from puberty to adulthood in mouse testis. Immunostaining results confirmed the localization of CCR 6 in testis. Further chemotaxis assays demonstrated that spermatogenic cells GC-1 and −2 exhibited a directional movement toward CCR6-specific ligand such as CCL20 or Sertoli cells in vitro. Conclusions The present findings indicate that CCR6 is involved in the chemotaxis of spermatogenic cells in vitro and promotes chemotaxis under non-inflammatory conditions during normal spermatogenesis.