This study aimed to evaluate the effects of partial reducing rumen-protected Lys (RPLys) on rumen fermentation and microbial composition in heifers. Three ruminal fistulated Holstein Friesian bulls were used to determine the effective degradability of RPLys using an in situ method at incubation times of 0, 2, 6, 12, 16, 24, 36, and 48 h. Thereafter, 36 Holstein heifers at 90 days of age were assigned to one of two dietary treatments: a theoretically balanced amino acid diet (PC group; 1.21% Lys, 0.4% Met) or a 30% Lys-reduced diet (PCLys group, 0.85% Lys, 0.4% Met). Rumen fluid samples from five heifers in each group were extracted using esophageal tubing on day 90 to determine pH, microprotein, ammonia, volatile fatty acids, and microbial communities. Results showed that the effective ruminal degradability was 25.76%. Furthermore, differences in rumen fermentation parameters and alpha diversity of the microbiota between the two groups were not significant, but beta diversity was significant. Based upon relative abundance analysis, short-chain fatty acid-producing bacteria, includingSharpea,Syntrophococcus,[Ruminococcus]_gauvreauii_group,Acetitomaculum, and[Eubacterium]_nadotum_groupbelonging toFirmicutes, were significantly decreased in the PCLys group. Spearman's analysis revealed a positive correlation between the butyrate molar proportion and the relative abundance of butyrate-producing bacteria such as[Eubacterium]_nadotum_group,Coprococcus_1,Ruminococcaceae_UCG_013,Pseudoramibacter, andLachnospiraceae_UCG_010. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States analysis further validated that RPLys deduction influenced energy metabolism. Together, our findings highlight the role of RPLys or Lys in butyrate-producing bacteria. However, the number of bacteria affected by Lys was very limited and insufficient to alter rumen fermentation.