In plants, there is an active prezygotic interspecific-incompatibility mechanism to prevent unfavorable hybrids between two species. We previously reported that an uncharacterized protein with four-transmembrane domains, named as Stigmatic Privacy 1 (SPRI1), is responsible for rejecting heterospecific pollen grains in Arabidopsis thaliana. However, the lack of notable functional domains in SPRI1 has limited our understanding of its biochemical properties. In this study, we conducted a functional analysis of the SPRI1 protein through point-mutational experiments and biochemical analysis. We explored the molecular regulatory mechanisms of SPRI1 and the relationships with its function. Alanine- and glycine-scanning experiments together with the evolutional analysis showed that the structural integrity of the C-terminal regions of the extracellular domain of this protein is important for its function. In addition, we found two cysteines (C67 and C80) within the extracellular domain that may be involved in the formation of intermolecular disulfide bonds. These cysteine residues are required for the stabilization of the SPR1 protein. Furthermore, SPRI1 may form homo-multimers and is present as part of a ∼300 kDa complex. Our present study indicates that SPRI1 forms large protein machinery for the rejection of hetero-specific pollen in stigmatic papilla cells.