To examine tight junction protein abundance and apoptosis of epithelial cells at the onset of involution in rodent mammary glands, milk accumulation and mammary engorgement were induced by teat‐sealing with an adhesive for 0, 6, 12, 18, 24, and 36 h (n = 6 per group) at peak lactation. In non‐sealed control glands, histological analysis confirmed a lactating phenotype, indicating suckling by pups throughout the experiment. In contrast, alveoli of teat‐sealed glands were distended within 6 h, with maximal luminal size observed by 12 h of non‐suckling. By 18 h following teat‐sealing, an involuting phenotype was observed, indicated by alveolar lumina engorged with milk vesicles and increased leukocytes. Relative to non‐sealed glands, mammary apoptosis was increased in engorged glands 18 h following teat‐sealing. The abundance of ZO‐1 and occludin proteins was decreased in engorged glands by 12 and 18 h, respectively, following teat‐sealing. In contrast, the claudin‐1 22 kDa band was increased by 6 h and peaked at 12–18 h, whereas the 28 kDa band declined by 36 h, relative to controls. There were no temporal changes in ZO‐1, occludin, and claudin‐1 22 kDa proteins within control glands, although there were minor differences in claudin‐1 28 kDa. These data indicate that intramammary milk accumulation due to cessation of milk removal is associated with mammary apoptosis. The apoptotic event is preceded by a rapid loss of abundance of ZO‐1, occludin and an initial increase in claudin‐1. The loss of cell–cell communication may initiate involution and apoptosis of mammary epithelial cells and is a localized intramammary event, occurring only in non‐suckled glands. J. Cell. Physiol. 232: 2075–2082, 2017. © 2016 Wiley Periodicals, Inc. To examine tight junction protein abundance and apoptosis of epithelial cells at the onset of involution in rodent mammary glands, milk accumulation and mammary engorgement was induced by teat‐sealing for 0, 6, 12, 18, 24, and 36 h (n = 6 per group) at peak lactation. The loss of cell–cell communication may initiate involution and apoptosis of mammary epithelial cells and is a localized intramammary event, occurring only in non‐suckled glands.