The current study was performed to clarify the effects of GHRH treatment on milk production and plasma hormones and metabolites in lactating Japanese Black cows (a beef breed) under negative energy balance (EB). Ten multiparous lactating beef cows were offered a normal-energy diet daily (110% of ME requirements for maintenance and lactation) until 5 d in milk (DIM) to standardize the cows before dietary treatment. From 6 DIM to the final days (63 DIM) of the experiment, the cows were allotted to experimental dietary treatments: 5 cows were offered a diet formulated for 130% [high-energy diet (HED)] and the remaining 5 cows were offered a diet formulated for 80% [low-energy diet (LED)] of ME requirements for maintenance and lactation. In addition, all cows received daily subcutaneous injections of 3 mg of bovine GHRH from 36 to 56 DIM (GHRH treatment period). Differences in BW of HED- and LED-fed cows at 63 DIM were +28.4 and -7.2 kg compared with BW at 6 DIM, and HED- and LED-fed cows were under positive EB (+23.7 MJ/d) and negative EB (-11.6 MJ/d) throughout the experiment period. Treatment with GHRH increased (P<0.01) the average daily milk yield to 6.2 kg in HED-fed cows compared with a milk yield of 5.3 kg for 7 d before the GHRH treatment period (pretreatment period); LED-fed cows had no increase in milk production from GHRH treatment. Plasma GH, IGF-1, insulin, and glucose concentrations increased (P<0.05) after GHRH treatment in both HED- and LED-fed cows; GHRH treatment also induced an increase (P<0.05) in the net area under the curve of plasma insulin after glucose challenge in both HED- and LED-fed cows. Plasma urea N concentrations were decreased (P<0.05) by GHRH treatment in HED-fed cows, but not in LED-fed cows. Plasma NEFA concentration was unaffected by GHRH treatment in both HED- and LED-fed cows. We conclude that GHRH treatment of lactating Japanese Black cows stimulates endogenous GH and subsequent IGF-1 secretion and might induce an increase in insulin resistance, irrespective of EB; however, compared with lactating dairy cows, both galactopoietic and lipolytic effects of GHRH might be insufficiently exerted under negative EB in lactating beef cows.