This study aimed to determine the effects of replacing corn with hydroponically sprouted barley (HSB) in Holstein heifer feed on growth performance and blood metabolites. For this purpose, the barley was prepared with hydroponic sprouting under optimized conditions, and its chemical composition was analyzed on a dry matter (DM) basis. A total of 30 Holstein heifers that were 7 months old with an average body weight of 212.60 ± 55.00 kg were used in a completely randomized design comprising 10 repetitions and three groups over a period of 11 months. Dietary treatments included control (CON), basal diet (corn meal-based diets), and trail diets, in which 10% and 30% HSB gradually replaced corn meal. The growth performance and biochemical indices were analyzed every month. Results showed that the HSB diet increased chemical compositions compared with the control diets on a DM basis. In addition, diets with up to 30% HSB did not show any negative effects on the growth performance (body weight, height at withers, and height at rump) or selected blood metabolites of heifers compared with the CON diet. In conclusion, the use of HSB instead of corn meal did not lead to any adverse effects on growth performance or biochemical indices; therefore, HSB can be used as an alternative dietary source for raising Holstein heifers.