In the previous paper, we reported that four proteins of 100kDa, 80kDa, 68kDa and 50kDa from the mouse brain interacted with estrogen receptor (ER) α-transactivation domain (TAD) and 68kDa protein showed age and sex dependent changes. Here, we describe the identification of 50kDa protein as β-tubulin and changes in its interaction with age and sex in mouse brain. It is a microtubule-associated protein which binds to activation function (AF)-1 region of ERα-TAD and is involved in estrogen signaling. The extent of interaction of mouse ERα-TAD with β-tubulin was higher in adult female as compared to old female and adult male. However, the expression of β-tubulin showed no significant change with age and sex. Such age and sex dependent alteration in the interaction of β-tubulin might account for the estrogen-mediated brain functions during aging.