The incidence of bovine mastitis caused by K. pneumoniae increases during summer. This experiment aimed to clarify the connection between heat stress and K. pneumoniae-induced mastitis in terms of inflammatory responses and barrier function. From day 9-16 of the postpartum period, lactating rats were exposed to 35 °C in the daytime and 29 °C at night to establish a heat stress model. On day 8 of the heat stress treatment, the rat mastitis model was established by intramammary infection with K. pneumoniae through the milk ducts. At 12 h post-infection, the rectal temperature, serum biochemistry, K. pneumoniae burden, as well as histopathology, epithelium integrity, inflammatory response, and HSP70 expression of the mammary glands were detected. The results showed that heat-stressed rats with K. pneumoniae infection displayed higher rectal temperatures, more neutrophil infiltration, and more significant pathological damage to the acinar lumen. Heat stress promoted the disruption of the structural integrity of tight junction and the downregulation of relative protein expressions (ZO-1, Occludin, and Claudin-3) as a consequence of the increased production of inflammatory parameters (endotoxin, MPO, IL-1β, IL-6, and TNF-α) by accelerating NF-κB pathway activation and HSP70 expression after K. pneumoniae infection. Thus, heat stress disrupts mammary epithelium integrity, contributing to the pathogen invasion, and aggravates intramammary damage during K. pneumoniae mastitis by facilitating barrier disruption and inflammatory response, which could trigger more severe mastitis.