Previously, we reported that GlyCAM 1 was a novel target for PRL (PRL) in the mouse mammary gland. However, the signaling pathway by which PRL regulates GlyCAM 1 expression has not been specified. In the present study, we showed that PRL induced GlyCAM 1 expression in primary mammary epithelial cells (PMEC) of mice through the Jak2/Stat5 pathway. Deletion and site-directed mutagenesis analyses of the GlyCAM 1 promoter demonstrated that the two tandemly linked Stat5 binding sites (GAS1 and GAS2) in the proximal promoter region were crucial and synergistically responded to PRL. GAS2, a consensus GAS site, was essential, and by itself weakly responded to PRL, whereas GAS1, a non-consensus site, failed to respond to PRL, but was indispensable for the maximal activity of the GlyCAM 1 promoter. Gel shift assays showed that probe containing GAS1 and GAS2 bound two Stat5 complexes, which represent Stat5 dimer and tetramer respectively, while GAS2 by itself bound Stat5 as a dimer only, and GAS1 showed no apparent binding activity. Interruption of tetramer formation by mutation of a tryptophan to alanine (W37A), and a leucine to serine (L83S) in the N terminus of Stat5A attenuated the synergistic effect between the two tandemly linked GAS sites. Overexpression of W37A and L83S mutants in PMEC suppressed endogenous GlyCAM 1 expression.