[Objective] PEX (Pollen extensin-like) gene is involved in controlling pollen development，which plays a crucial role in the embryogenesis of pollen microspores and haploid culture. In this study, we isolated the CaPEX3 gene from pepper and analyzed its protein structure and expression pattern, which provides a theoretical foundation for further investigation into the function of the CaPEX3 gene. [Methods] CaPEX3 was cloned by PCR, and its protein structure, evolution and gene regulation were analyzed by bioinformatics. The expression of CaPEX3 in different tissues and pollens of different varieties were detected by qRT-PCR. The pollen viabilities of different varieties of pepper were detected by staining method and their relationships with the expression of CaPEX3 were analyzed. [Results] The coding region of CaPEX3 was 1 581 bp in length, encoding 526 amino acids, which belongs to the LRX (Leucine-rich Repeats/Extensins) gene family. The protein has phosphorylation, glycosylation sites, signaling peptides and transmembrane domains. The expression level of CaPEX3 was the highest in flowers, and the gene expression was higher in tissues with high pollen vitality. The regulatory network predicted that CaPEX3 may be involved in the regulation of pollen development through the action of genes related to flower development. [Conclusion] The expression of CaPEX3 was specifically localized to flowers, and a correlation was observed between its expression level and the activity of pollen grains. The analysis of gene expression and the construction of related vectors provide a theoretical foundation and technical support for the subsequent investigation of gene function and mechanism of CaPEX3.