Environmental and nutritional factors influence productivity of dairy cows during lactation, possibly via similar physiological mechanisms. The objective of this study was to test the effects of heat stress (HS) and metabolizable protein (MP) on milk production and plasma metabolites. Thirty-six multiparous Holstein cows (219 ± 27 d in milk) were assigned to a factorial arrangement of treatments in a completely randomized design. Treatments were imposed for 21 d and consisted of HS or cooling with fans and sprinklers (CO) and high or low MP diets [17.9% (HMP) or 14.2% (LMP) crude protein on a DM basis]. Cows were exposed to prevailing weather of July and August in Tennessee. By design, CO cows were feed restricted to match intake of HS cows receiving the LMP treatment. Body temperature was monitored, and milk and blood samples were collected and analyzed. Treatment effects were tested using the Mixed procedure in SAS (LSM ± SEM). No interactions were detected, thus main effect differences are reported hereafter. Compared with CO, HS increased (P ≤ 0.03) rectal (38.4 vs. 39.0 ± 0.09°C) and vaginal temperatures (38.6 vs. 39.3 ± 0.09°C) and respiration rate (48.6 vs. 76.2 ± 2.2 breaths/min). Relative to CO, HS decreased (P < 0.01) yields of energy-corrected milk (37.5 vs. 33.9 ± 0.8 kg/d), protein (0.99 vs. 0.93 ± 0.02 kg/d), and fat (1.47 vs. 1.30 ± 0.04 kg/d) but did not affect circulating insulin (21.4 vs. 21.7 ± 3.02 µU/mL). Metabolizable protein did not affect energy-corrected milk yield. Low MP increased (P ≤ 0.05) milk fat yield (1.36 vs. 1.41 ± 0.04 kg/d) and plasma free fatty acid concentrations (159 vs. 204 ± 21.6 µEq/L) but decreased (P < 0.01) circulating insulin (28.3 vs. 14.9 ± 3.0 µU/mL). In summary, HS did not affect circulating insulin and reduced synthesis of protein, fat, and energy-corrected milk. Low MP reduced circulating insulin, increased circulating free fatty acids, and did not affect energy-corrected milk yield. Hormonal and metabolite adaptations in LMP cows may have promoted the use of nutrients to support milk synthesis.