Hepatocellular carcinoma (HCC) is a formidable malignancy, with growing interest in identifying cancer stem cells (CSCs) as potential therapeutic targets. CD44 isoforms have emerged as promising CSC markers in HCC, often associated with epithelial-mesenchymal transition (EMT) induced by transforming growth factor-beta (TGF-β). However, the intricate relationship between CSC traits, CD44 isoforms, and TGF-β effects on CD44 subpopulations in HCC remains unclear. This study aimed to clarify how TGF-β influences proteomic changes and CSC traits in subpopulations expressing standard CD44 isoform (CD44std) and CD44 variant 9 (CD44v9). Treating SNU-423 cells with TGF-β lead to notable morphological changes, resembling a spindle-like phenotype, along with reductions in CD44v9+ subpopulations and differential CD44std expression. Proteomic analysis highlighted significant alterations in signaling pathways, particularly the mitogen-activated protein kinase (MAPK) pathway. Validation experiments demonstrated upregulation in CD44std cells and downregulation in CD44v9 cells post-TGF-β treatment. Furthermore, TGF-β exerted regulatory influence over Sox2 and Nanog expression, resulting in increased colony and spheroid formation in CD44std cells but decreased capabilities in CD44v9 cells. TGF-β also enhanced the migratory and invasive properties of both subpopulations through EMT, alongside increased adhesive abilities in CD44v9 cells. These findings illuminate the dynamic interplay between TGF-β and CD44std/CD44v9 subpopulations, emphasizing the role of MAPK signaling and modulation of CSC traits. This research contributes to understanding the dynamic interplay between CD44 isoforms and TGF-β in HCC.