Tetratricopeptide repeat domain 9A (TTC9A) belongs to a family of TTC9 proteins. Its induction by progesterone in breast cancer cells was associated with marked growth inhibition and induction of focal adhesion. TTC9A interacts specifically with actin-binding protein tropomyosin Tm5NM-1 which stabilizes actin filament and focal adhesion. However, the function of TTC9A is still obscure. This study exploited mice model to characterize the regulation of TTC9A gene expression during mammary development and explored possible mechanisms of TTC9A gene regulation. It was demonstrated that mammary TTC9A expression is distinctively down-regulated in gland undergoing functional differentiation (lactation) and up-regulated during involution. Furthermore, TTC9A expression during lactation and involution is regulated by the factors in the local microenvironment. This is illustrated with teat sealing model in which the teat sealed glands (undergoing involution) expressed significantly higher levels of TTC9A protein and mRNA than the contralateral non-sealed lactating glands. Importantly, this local induction of TTC9A expression upon involution coincided with the re-activation of estrogen receptor α (ERα). Together with the observation that TTC9A is a direct ERα target gene, we propose that the fall and rise of TTC9A levels during lactation and involution is caused by the changes of ERα activity that is in turn regulated by the factors in the microenvironment.