Our objective was to determine the effects on milk production and rumen fermentation of feeding cows hydroponically grown barley sprouts (HydroGreen Inc., Renner, SD). Twenty mid-lactation Holsteins (DIM 205 ± 47.4) were used in a 6-wk randomized complete block design study. Treatments included: 1) control diet with ground corn and soybean meal as major concentrate ingredients (CON) and 2) 8% (DM basis) as hydroponic barley sprouts replacing some corn and soybean meal (HYD). Both diets were individually fed as total mixed rations using Calan gates and were isocaloric and isonitrogenous. Cows were milked 2 × /d. Body condition scores (BCS) and body weight (BW) were measured and rumen fluid was collected via esophogeal tube at 4 h post feeding on 2 d during wk 0, 2, 4, and 6. Milk samples were taken on the same days. A blind triangle taste test was done on 2 d in wk 6 with 25 volunteers. Data were analyzed using MIXED procedures of SAS 9.4 with repeated measures and the triangle test was analyzed using a Chi-squared test in R. Dry matter intake (23.0, and 23.0 kg/d, for CON and HYD, respectively; SEM = 0.54) was similar (P > 0.05) between treatments. Treatments had similar BW (690.0, and 680.5 kg; SEM = 3.72). Body condition score (3.09, and 3.08; SEM = 0.03) was greater (P = 0.03) for CON fed cows. Milk production (30.6, and 31.5 kg/d; SEM = 1.60) and feed efficiency (1.33 and 1.40; SEM = 0.046) had treatment by wk interactions (P = 0.01). Milk protein (0.94 and 0.94 kg/d; SEM = 0.03) and fat (1.10 and 1.07, SEM = 0.06) yields were similar (P > 0.05). Rumen pH (6.53, and 6.60; SEM = 0.11), ammonia-N (12.72, and 13.90 mg/dL; SEM = 1.00), total volatile fatty acids (95.6, and 95.0 mM; SEM = 1.81) and acetate to propionate ratio (3.0, and 3.1; SEM = 0.11) were similar. Triangle test participants were unable to discern a taste difference between milk from CON vs. HYD on d 1 (Chi-squared = 0.55; P = 0.46) and d 2 (Chi-squared = 1.67; P = 0.20). Results demonstrate that hydroponic barley sprouts can replace a portion of the corn and soybean meal and maintain rumen fermentation and lactation performance.