Epidemiological, cellular, and genetic analyses indicate the hormone prolactin (PRL) and its cognate receptor in humans (hPRLr) are significantly involved in breast cancer pathogenesis. Recent evidence demonstrated that a truncated mouse PRLr (mPRLrT) is oncogenic when expressed with canonical long mPRLr (mPRLrL). mPRLrT shares significant sequence homology with a naturally-occurring and widely-expressed hPRLr isoform, the intermediate hPRLr (hPRLrI). As determined by tissue microarray (TMA), hPRLrI is expressed in >85% of breast cancer, with expression increasing as a function of both tumor grade and Ki67 status. To confirm the oncogenic potential of hPRLrI, isoform-specific hPRLrI knock-down (KD) was performed in breast cancer cell line MCF7. hPRLrI KD resulted in a significant decrease in proliferation, migration, and anchorage-independent growth. Given the homology between mPRLrT and hPRLrI, we hypothesized hPRLrI may similarly induce transformation, when expressed alongside wild-type long hPRLr (hPRLrL). hPRLrL/I co-expression in the immortalized but not transformed human breast cell line MCF10A resulted in a significant increase in proliferation, migration, and anchorage-independent growth. These results were not observed following overexpression of either isoform alone, demonstrating that hPRLrL/I co-expression is necessary to induce transformation of normal mammary epithelia. To test our hypothesis in vivo, we established MCF10A xenografts using female NSG mice. Following intraductal injection, we observed rapid tumor growth in the hPRLrL/I cohort, significantly over that of expressing either isoform alone. To determine mechanisms of transformation, we examined both differential protein stability and altered signaling events. In analyzing receptor degradation, a cycloheximide assay revealed hPRLrL stability is increased when heterodimerized with hPRLrI. hPRLrL turnover is impaired in breast cancer, indicating this phenomenon may be involved in the observed hPRLrI-mediated transformation. Regarding differential signaling, we examined the Jak2/Stat5a pathway. Jak2 is a promiscuous kinase whose oncogenic actions are well-characterized, while Stat5a is a transcription factor whose activities are critical in attenuating the oncogenicity of Jak2. Following PRL stimulation, it was observed that hPRLrL/I co-expression induced approximately two-fold greater Jak2-Y1007/1008 phosphorylation (pJak2) compared to that induced by hPRLrL expression alone. Further, it was observed that hPRLrL/I co-expression induced ten-fold less Stat5a-Y694 phosphorylation (pY-Stat5a) than hPRLrL expression alone. These data indicate unchecked pJak2 activity may also be a contributing mechanism in the observed transformation. Overall, these results demonstrate that hPRLrI, alongside hPRLrL, is sufficient for transformation of normal breast tissue.