The objective of the study was to examine the effect of the dietary inclusion of hydroponic barley grass (HBG) on the performance of mid-lactation dairy cows when fed 2 different protein levels. Forty-eight Holstein dairy cows that were, 119 ± 14.8 d in milk and yielding 39.9 ± 4.7 kg/d milk were randomly allocated to one of 4 treatment groups (n = 12) in a 2 × 2 factorial arrangement. Cows were fed a control diet without HBG (HC; 16.0% CP), a low protein diet without HBG (LC; 14.5% CP), a control diet with HBG (HB; 16.0% CP), or a low protein diet with HBG (LB; 14.5% CP). The HBG replaced 2.25 kg DM of alfalfa hay in treatments HB and LB. Cows were fed the diets as a TMR twice daily using a Calan Broadbent Individual Animal Feeding System. The experimental period consisted of 1 wk for adaptation to the diet and 8 wks for sampling. Individual intake was recorded daily and feed samples collected 3 time a week for subsequent analysis of their nutrient content. Milk yield was recorded daily with samples collected weekly from 3 consecutive milkings and analyzed for composition. Data were analyzed using the PROC MIXED of SAS. Protein level, HBG, and the 2-way interaction between HBG and protein level were the fix effects in the model. The HBG and dietary protein level did not affect DMI, milk yield or energy corrected milk yield (P > 0.05), with mean values of 24.6, 36.5 and 36.5 kg/d respectively. Similarly, milk composition was not affected by the inclusion of HBG or dietary protein level (P > 0.05), and there no effect of treatment on feed conversion efficiency or N utilization (P > 0.05). However, there was a trend for an interaction (P = 0.05) between HBG and dietary protein level, with cows fed HBG and 16.0% CP having the lowest cost of production at 1.74 yuan/kg milk.