The estrogen receptor α (ERα) is a transcription factor that mediates the biological effects of 17β-estradiol (E2). ERα transcriptional activity is also regulated by cytoplasmic signaling cascades. Here, several Gα protein subunits were tested for their ability to regulate ERα activity. Reporter assays revealed that overexpression of a constitutively active Gαo protein subunit potentiated ERα activity in the absence and presence of E2. Transient transfection of the human breast cancer cell line MCF-7 showed that Gαo augments the transcription of several ERα-regulated genes. Western blots of HEK293T cells transfected with ER±Gαo revealed that Gαo stimulated phosphorylation of ERK 1/2 and subsequently increased the phosphorylation of ERα on serine 118. In summary, our results show that Gαo, through activation of the MAPK pathway, plays a role in the regulation of ERα activity.