To study the biological significance and function of sperm flagellar protein 1 (Spef1) in the formation and assembly of spermatozoa, rapid amplification of cDNA ends (RACE) and qRT-PCR techniques were used to clone the gene cDNA of the Spef1 in Sepiella japonica and analyse the tissue expression specificity. The results showed that a 1 135 bp full-length cDNA of Spef1 gene from S. japonica (described as SjSpef1) was obtained, which consisted of a 178 bp 5' untranslated region (UTR) and a 165 bp 3'UTR. The predicted open reading frame (ORF) was 792 bp, molecular weight of the deduced protein was 30.567 7 ku, and its pI was 7.03. The SjSpef1 was hydrophilic, without signal peptide sequence and transmembrane regions, and it might play a intracellular role. Secondary structure analysis revealed that it contained rich spiral structures (49%). Homology modeling indicated that CH2 domain of Spef1 mainly consisted of four spiral structures, which were connected by some loop structures. The multiple sequence alignment showed that SjSpef1 had the highest similarity with Octopus bimaculoides which was 59.49%, which showed that Spef1 was not very conserved in evolution. Phylogenetics analysis based on Spef1 demonstrated that S. japonica had the closest relationship with O. bimaculoides. SjSpef1 gene was expressed significantly in testis by qRT-PCR. Its cloning and analysis of tissue specific expression will be of great significance to exploring its cellular localization and biological function in the future.