Water holding capacity (WHC) is an important sensory attribute that greatly influences meat quality. However, the molecular mechanism that regulates the beef WHC remains to be elucidated. In this study, the longissimus dorsi (LD) muscles of 49 Chinese Simmental beef cattle were subjected to RNA sequencing (RNA-seq), among which eight individuals with the highest WHC (H-WHC) and the lowest WHC (L-WHC) were selected for transcriptome analysis. A total of 1256 genes were identified as differentially expressed genes (DEGs) between two groups, of which 948 genes were up-regulated and 308 genes were down-regulated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment revealed that DEGs were significantly enriched in 24 GO terms and 78 pathways. Additionally, based on protein-protein interaction (PPI) network, animal QTL database (QTLdb), and relevant literature, the study not only confirmed seven genes (HSPA12A, HSPA13, PPARg, MYH10, MYL2, MYPN, and TPI1) influenced WHC in accordance with previous studies, but also identified six genes (ITGAV, FGF2, THBS1, DCN, COL4A1, and TGFBR1) as the most promising novel candidate genes affecting the WHC. These findings could offer important insight for exploring the molecular mechanism underlying the WHC trait and facilitate the improvement of beef quality.