The objective of this study was to construct a mammary gland specific gene targeting vector (pLG), which could help the exogenous goat growth hormone (GH) cDNA being integrated into the beta-casein locus of goats and improve the milk production of goat. We cloned the 5' and 3' regulatory sequences of goat's beta-casein gene as homologous arms and inserted them into the Not I/Xho I and Sal I/Cla I multiple clone sites (MCS) of pLoxp II vector respectively, then inserted the GH cDNA into the Xho I MCS of pLoxp II vector and finally got the gene targeting vector pLG. The expression of GH in Bcap-37 cells transfected with plasmid pLG (14.52 ng/mL) was two times higher than that of cells untreated (7.10 ng/mL). While the GH expression of goats injected with plasmid pLG (7.54 ng/mL) was higher than goats untreated or injected with normal saline (6.64 and 6.78 ng/mL). We successfully constructed the mammary gland specific beta-casein gene targeting vector pLG exhibiting bioactivity of transcription and expression of GH both in vitro and in vivo.