This study aimed to assess the effects of partially substituting soybean meal in the diet with slow-release urea (SRU) on the lactation performance, heat shock signal molecules, and environmental sustainability of heat-stressed lactating cows in the middle stage of lactation. In this study, 30 healthy Holstein lactating dairy cattle with a similar milk yield of 22.8 ± 3.3 kg, days in milk of 191.14 ± 27.24 days, and 2.2 ± 1.5 parity were selected and randomly allocated into two groups. The constituents of the two treatments were (1) basic diet plus 500 g soybean meal (SM) for the SM group and (2) basic diet plus 100 g slow-release urea and 400 g corn silage for the SRU group. The average temperature humidity index (THI) during the experiment was 84.47, with an average THI of >78 from day 1 to day 28, indicating the cow experienced moderate heat stress conditions. Compared with the SM group, the SRU group showed decreasing body temperature and respiratory rate trends at 20:00 (p < 0.1). The substitution of SM with SRU resulted in an increasing trend in milk yield, with a significant increase of 7.36% compared to the SM group (p < 0.1). Compared to the SM group, AST, ALT, and γ-GT content levels were significantly increased (p < 0.05). Notably, the levels of HSP-70 and HSP-90α were significantly reduced (p < 0.05). The SRU group showed significantly increased acetate and isovalerate concentrations compared with the SM group (p < 0.05). The prediction results indicate that the SRU group exhibits a significant decrease in methane (CH₄) emissions when producing 1 L of milk compared to the SM group (p < 0.05). In summary, dietary supplementation with SRU tended to increase the milk yield and rumen fermentation and reduce plasma heat shock molecules in mid-lactation, heat-stressed dairy cows. In the hot summer, using SRU instead of some soybean meal in the diet alleviates the heat stress of dairy cows and reduces the production of CH₄.