Smoking induces a decrease in breast milk volume, adverse changes in milk composition, and a shorter lactation period in breastfeeding women. In breastfeeding women, nicotine from tobacco is transferred from the blood to breast milk. Previously, we reported that nicotine adversely affects milk production and tight junctions (TJs) in mammary epithelial cells (MECs) in vitro. However, the mechanisms by which nicotine influences milk production and TJs in MECs remain unclear. During lactation, MECs are in contact with acetylcholine (ACh) in milk and express multiple nicotinic ACh receptors (nAChRs). In this study, we investigated whether nAChRs and ACh are involved in milk production TJs in MECs using a culture model of MECs that exhibit milk production ability and formation of less-permeable TJs. The results showed that nAChRα2 and nAChRα3 agonists, Br-PBTC and NS3861, respectively, suppressed casein secretion and increased claudin-4, a TJ protein. In addition, Br-PBTC and NS3861 inactivated STAT5 and Akt, which are signaling molecules that facilitate milk production in MECs. However, ACh did not influence casein secretion, claudin expression, or the activation of STAT5 and Akt in MECs. In contrast, the acetylcholinesterase inhibitor (donepezil) and nAChRα3 antagonist (α-conotoxin PIA) inhibited casein secretion concurrently inactivating STAT5 and Akt. Furthermore, short-term treatment with Br-PDTC and NS3861 on the apical side of MECs induced the inactivation of STAT5 and Akt. These findings indicate that MECs regulate milk production and TJ formation by regulating the acetylcholine levels in milk and that nicotine adversely affect milk production in MEC by disrupting the ACh/nAChR axis.