Abstract: Sterols are major components of the eukaryotic membrane and play important roles in development processes. CYCLOPROPYL STEROL ISOMERASE1 (CPI1) encodes a plant-specific key enzyme in the plant sterol biosynthesis pathway. Only the CPI1 from Arabidopsis has been cloned and characterized. In this study, we identified single-copy CPI1 genes from algae to flowering plants. All CPI1 genes from land plants have a conserved gene structure and high protein sequence similarity ranging from 48% to 90%, with low protein sequence similarity between land plants and algae. Protein structure prediction revealed that all CPI1 proteins have a similar protein structure, with 7 transmembrane domains and 6 hydrophilic loops. RT-PCR revealed that the land plant CPI1 genes were constitutive expression genes. To investigate the function of CPI1 genes, CPI1 from Brachypodium distachyon was cloned and transformed into the Arabidopsis cpi1-1 mutant. The BdCPI1 gene could completely complement the function of AtCPI. Thus, our data for single copy number, conserved gene structures, protein structures, and gene expression patterns suggest that the functions of land plant CPI1 genes are highly conserved.