Gametophytic self-incompatibility (GSI) is an important factor that can bring out seedless fruit in Citrus，GSI was regulated by a highly polymorphic locus named the S-locus. S-protein Homologous (SPH) gene belonged to the S-locus gene, but the role of SPH gene in the reaction of self-incompatibility has not been reported. In the study, the CDS of SPH gene, named CrSPH, was cloned from ‘Wuzishatangju’ and ‘Shatangju’. the CDS was 417 bp encoding a protein of 138 amino acids. There were three base pairs difference between ‘Wuzishatangju’ and ‘Shatangju’, and two amino acids ware substituted between ‘Wuzishatangju’ and ‘Shatangju’. The Southern hybridization experiment showed there was one copy of CrSPH in the gene genomic DNA both Wuzishatangju’ and ‘Shatangju’.The qPCR showed that the expression level of CrSPH gene was significantly difference in flower organs of ''Shatangju'' , the highest expression level was found in ovary, and it was hardly expressed in petals, filaments, anthers, stigmas, styles. the relative expression level of CrSPH gene was 16 times higher in the ovaries of ‘Shatangju’ than that in ovaries of ‘Wuzishatangju’. CrSPH gene began to express at 0 d after cross-pollination (‘Wuzishatangju’× ‘Shatangju’), the expression level showed an upward trend from 2th to 4th day, the highest expression level was detected at the 6th day in pistil. The expression level was in pistil first up and then down after self-pollination (‘Wuzishatangju’בWuzishatangju’), but the expression level of self-pollination was far lower than that of cross-pollination. The prokaryotic expression experiment successfully induced the CrSPH heterologous protein, and the pollen germination experiment in vitro showed that the germination rate of ‘Wuzishatangju’ was significantly inhibited with increasing CrSPH protein concentrations from ‘Wuzishatangju’.