The dairy industry is facing challenges in balancing forage supply and crop production. Therefore, forage supply based on a farm land-saving approach should be developed to overcome the human–livestock competition on farmland. The objective of this study was to learn the potential impact of partially replacing oat hay with whole-plant hydroponic barley seedlings (HBS) produced via a land-saving hydroponic method on growth performance, digestibility, and rumen microbiota in Holstein dairy heifers. In total, 39 Holstein heifers were randomly divided into 13 blocks based on age and body weight for an 8-week experimental period. The heifers within each block were randomly allocated to one of three diets group: (1) 0% HBS and 16% oat hay (CON); (2) 4% HBS and 12% oat hay (25%HBS); and (3) 8% HBS and 8% oat hay (50%HBS). Compared to CON, feed intake, growth performance, and body N retention were similar to those in cows fed 25%HBS but lower in 50%HBS-fed animals (p < 0.05). Reduced digestibility (crude protein (CP) and organic matter (OM)) was observed in 50%HBS animals (p < 0.05). Compared to the control, the levels of Lachnospiraceae_XPB1014_group, Bacillus, and Colidextribacter were higher, but the levels of Sphaerochaeta and Ruminiclostridium were lower in 50%HBS animals (p < 0.05). Additionally, the digestibility of CP (p < 0.01, r = −0.61) and ether extract (EE) (p < 0.01, r = −0.58) was negatively correlated with Lachnospiraceae_XPB1014_group. The digestibility of OM (p = 0.01, r = −0.55), neutral detergent fiber (NDF) (p = 0.01, r = −0.56), acid detergent fiber (ADF) (p = 0.02, r = −0.52), and CP (p < 0.01, r = −0.61) was negatively correlated with Bacillus. The digestibility of NDF (p = 0.02, r = −0.52) and ADF (p = 0.03, r = −0.50) was negatively correlated with Colidextribacter. The digestibility of OM (p = 0.03, r = 0.50), NDF (p = 0.03, r = 0.50), and ADF (p = 0.03, r = 0.49) was positively correlated with Ruminiclostridium. The digestibility of OM (p = 0.04, r = 0.47), CP (p < 0.01, r = 0.58), and EE (p = 0.03, r = 0.49) was positively correlated with unclassified_f_Rikenellaceae. The digestibility of CP was positively correlated with Sphaerochaeta (p = 0.02, r = 0.53). In conclusion, the current study suggests that HBS could replace oat hay in a ratio-dependent manner. The reduced growth performance could be caused by lower feed intake and digestibility, which may be attributed to the alteration in the rumen’s microbial population. Further exploration of the inhibiting factors of HBS would broaden the application of hydroponic feed in the future.