The wild plant known as Calotropis procera is important in medicine, industry and ornamental fields. Due to spread in areas that suffer from environmental stress, it has a large number of tolerance genes to environmental stress such as drought and salinity. Proline is one of the most compatible solutes that accumulate widely in plants to tolerate unfavorable environmental conditions. Plant proline synthesis depends on Δ-pyrroline-5-carboxylate synthase (P5CS) gene. But information about this gene in C. procera is unavailable. In this study, we uncovered and characterized P5CS (P5CS, NCBI accession no. KJ020750) gene in this medicinal plant from the de novo assembled transcriptome contigs of the high-throughput sequencing dataset. A number of GenBank accessions for P5CS sequences were blasted with the recovered de novo assembled contigs. Homology modeling of the deduced amino acids (NCBI accession No. AHM25913) was further carried out using Swiss-Model, accessible via the EXPASY. Superimposition of C. procera P5CS-like full sequence model on Homo sapiens (P5CS_HUMAN, UniProt protein accession no. P54886) was constructed using RasMol and Deep-View programs. The functional domains of the novel P5CS amino acids sequence were identified from the NCBI conserved domain database (CDD) that provide insights into sequence structure/function relationships, as well as domain models imported from a number of external source databases (Pfam, SMART, COG, PRK, TIGRFAM).