Myrosinase is a defense-related enzyme and is capable of hydrolyzing glucosinolates into a variety of compounds, some of which are toxic to pathogens and herbivores. Many studies revealed that a number of important vegetables or oil crops contain the myrosinase-glucosinolate system. However, the related promoter and genomic DNA sequences as well as expression proifles of myrosinase gene remain largely unexplored in radish (Raphanus sativus). In this study, the 2798 bp genomic DNA sequence, designated asRsMyr2, was isolated and analyzed in radish. TheRsMyr2 consisting of 12 exons and 11 introns relfected the common gene structure of myrosinases. Using the genomic DNA walking approach, the 5′-lfanking region upstream ofRsMyr2 with length of 1711 bp was successfully isolated. PLACE and PlantCARE analyses revealed that this upstream region could be the promoter ofRsMyr2,which contained several basiccis-regulatory elements including TATA-box, CAAT-box and regulatory motifs responsive to defense and stresses. Furthermore, recombinant pET-RsMyr2protein separated by SDS-PAGE was identiifed as myrosinase with mass spectrometry. Real-time PCR analysis showed differential expression proifles ofRsMyr2 in leaf, stem and root at different developmental stages (e.g., higher expression in leaf at cotyledon stage and lower in lfesh root at mature stage). Additionally, theRsMyr2geneexhibited up-regulated expression when treated with abscisic acid (ABA), methyl jasmonate (MeJA) and hydrogen peroxide (H2O2), whereas it was down-regulated by wounding (WO) treatment. The ifndings indicated that the expression ofRsMyr2 gene was differentially regulated by these stress treatments. These results could provide new insight into elucidating the molecular characterization and biological function of myrosinase in radish.