Gametophytic self-incompatibility (GSI) is controlled by a highly polymorphic locus called the S-locus, which is an important factor that can result in seedless fruit in Citrus. The S₁ self-incompatibility locus-linked pollen 3.15 gene (S_1-3.15) belongs to a type of S locus gene. The role of S_1-3.15 in the SI reaction of Citrus has not yet been reported. In this study, full-length sequences of cDNA and DNA encoding the S_1-3.15 gene, referred to as CrS_1-3.15, were isolated from ‘Wuzishatangju’ (Self-incompatibility, SI) and ‘Shatangju’ (Self-compatibility, SC). The predicted amino acid sequences of CrS_1-3.15 between ‘Wuzishatangju’ and ‘Shatangju’ differ by only three amino acids. Compared to ‘Wuzishatangju’, three bases were substituted in the genomic DNA of CrS_1-3.15 from ‘Shatangju’. Southern blot results showed that one copy of CrS_1-3.15 existed in the genomic DNA of both ‘Wuzishatangju’ and ‘Shatangju’. The expression level of the CrS_1-3.15 gene in the ovaries of ‘Shatangju’ was approximately 60-fold higher than that in the ovaries of ‘Wuzishatangju’. When ‘Wuzishatangju’ was cross-pollinated, the expression of CrS_1-3.15 was upregulated in the ovaries at 3 d, and the highest expression levels were detected in the ovaries at 6 d after cross-pollination of ‘Wuzishatangju’ × ‘Shatangju’. To obtain the CrS_1-3.15 protein, the full-length cDNA of CrS_1-3.15 genes from ‘Wuzishatangju’ and ‘Shatangju’ was successfully expressed in Pichia pastoris. Pollen germination frequency of ‘Wuzishatangju’ was inhibited significantly with increasing CrS_1-3.15 protein concentrations from SI ‘Wuzishatangju’.