【Objective】Cloning and functional verification of Rat insulin 2 promoter （RIP2） were conducted to lay the foundation for breeding transgenic animals of specific expression target genes of insulin. 【Method】According to the RIP2 sequence （No. J00748.1） was published in GenBank， a pair of primer was designed to amplify the RIP2 sequence by PCR method. The RIP2 sequence was connected to the pMD18-T vector， the restructuring vector was doubly digested by Hind III and BamH I， and then the recycling RIP2 sequence was connected to pEGFP-1 vector to construct the pEGFP-1-RIP2 expression vector. The constructing vector was transfected into PK15 cells, and fluorescent protein expression in cells was observed after 24 hours. 【Result】The cloned RIP2 sequence was 703 bp （-871~-169 sites） in length and had 99.9% of homology the reference sequence （No. J00748.1）. There existed 3 base mutations， from initiation codon ATG upstream （+1）， G base deletion was at -57 site， C/A mutation at -387 site， G base insertion at -707 site. The RIP2 sequence contained 2 promoter elements: TATA-box（-206~-201 sites） and CAAT-box （-343~-339 sites）. Green fluorescence was observed from the PK15 cells transfected pEGFP-1-RIP2 plasmids after 24 hours. 【Conclusion】The cloned RIP2 sequence has promoter function， but its activity is weaker than CMV promoter.