Estrogen receptor α (ERα) functions as both a transcription factor and a mediator of rapid estrogen signaling. Recent studies have shown a role for ERα-interacting membranous and cytosolic proteins in ERα action, but our understanding of the role of the microtubule network in the modulation of ERα signaling remains unclear. Here we found that endogenous ERα associates with microtubules through the microtubule-binding protein hematopoietic PBX-interaction protein (HPIP). Biochemical and RNA-interference studies demonstrated that HPIP influences ERα-dependent rapid estrogen signaling by acting as a scaffold protein and recruits Src kinase and the p85 subunit of phosphatidylinositol 3-kinase to a complex with ERα, which in turn stimulates AKT and MAPK. We also found that ERα interacts with β-tubulin through HPIP. Destabilization of microtubules activated ERα signaling, whereas stabilization of microtubules repressed ERα transcriptional activity in a HPIP-dependent manner. These findings revealed a role for HPIP–microtubule complex in regulating 17β-estradiol–ERα responses in mammalian cells and discovered an inherent role of microtubules in the action of nuclear receptor.