To compare the regulation effects by different promoters on bovine prolactin gene expression in different cell lines, three recombinant bovine prolactin expression vectors were constructed using different promoters, i.e., CMV promoter, bovine prolactin gene promoter and goat β-casein gene promoter, respectively named pCMV, pPRLP and pP1A3, which were transfected into two cell lines, mouse pituitary tumor cell strain (AtT20) and mouse mammary epithelial cell strain (HC11), respectively. RT-PCR and real-time RT-PCR were used to investigate the expression level of the above three vectors in both cell lines. pCMV vector was effectively expressed in both cell lines, pPRLP vector had a similar expression level to that of pCMV in both cell lines, pP1A3 was expressed in HC11 but not in AtT20. pP1A3 was tissue-specific to mammary gland. pPRLP was able to express with a significant level in pituitary and mammary glands, while its tissue-specific characteristics in other tissues need further investigation.