The study investigated the effects of dietary protein level and the inclusion of hydroponic barley sprouts (HB) on lactation performance, blood biochemistry and N use efficiency in mid-lactation dairy cows. Treatments were arranged in a 2 × 2 factorial design with 2 crude protein (CP) levels [16.8% and 15.5% of dry matter (DM)], with HB (4.8% of DM, replacing 4.3% of alfalfa hay and 0.5% of distillers dried grains with solubles (DDGS)) or without HB. Forty-eight multiparous Holstein dairy cows (146 ± 15 d in milk, 40 ± 5 kg/d of milk) were randomly allocated to 1 of 4 diets: high protein diet (16.8% CP, HP), HP with HB (HP+HB), low protein diet (15.5% CP, LP), or LP with HB (LP+HB). An interaction between CP × HB on dry matter intake (DMI) was detected, with DMI being unaffected by HB inclusion in cows fed the high CP diets, but was lower in cows fed HB when the low CP diet was fed. A CP × HB interaction was also observed on milk and milk protein yield, which was higher in cows fed HB with HP, but not LP. Inclusion of HB also tended to reduce milk fat content, and feeding HP resulted in a higher milk protein and milk urea N content, but lower milk lactose content. Feed efficiency was increased by feeding HP or HB diets, whereas N efficiency was higher for cows fed LP or HB diets. There was an interaction on the apparent total-tract digestibility of DM and CP, which was higher when HB was fed along with HP, but reduced when fed with LP, whereas the digestibility of ADF was increased by feeding low protein diets. In conclusion, feeding a low protein diet had no adverse effect on cow performance, while feeding HB improved milk and milk component yield, and N efficiency when fed with a high CP diet, but compromised cow performance with a low CP diet.