In lactating mammary glands, alveolar mammary epithelial cells (MECs) synthesize and secrete milk components. MECs also form less permeable tight junctions (TJs) to prevent the leakage of milk components. During lactation, MECs are exposed to temperature changes by metabolic heat production and air ambient temperature. In this study, we investigated whether temperature changes influence milk production ability and TJ barriers in MECs by using two lactating culture models. The results showed that 39 °C treatment activated milk production and enhanced the formation of less-permeable TJs. In contrast, 41 °C treatment caused adverse effects on the TJ barrier and cell viability, although the milk production ability of MECs was temporarily up-regulated. MECs cultured at 37 °C showed relatively low milk production ability and high proliferation activity. Furthermore, we investigated three kinds of transcription factors relating to lactogenesis, signal transducer and activator of transcription 5 (STAT5), STAT3 and glucocorticoid receptor (GR). STAT5 signaling was activated at 39 and 41 °C by an increase in total STAT5. However, long-term treatment led to a decrease in total STAT5. STAT3 signaling was inactivated by high temperature treatment through a decrease in total STAT3 and inhibited phosphorylation of STAT3. GR signaling was continuously activated regardless of temperature. These results indicate that a moderate high temperature condition at 39 °C induces a high lactation capacity of MECs through control of STAT5 and STAT3 signaling. In contrast, long-term exposure at 41 °C leads to a decline in milk production capacity by inactivation of STAT5 and a decrease in the total number of MECs.