Rosmarinic acid (RA), isolated from herbal balm mint plants, has demonstrated potent anti-tumor properties against liver cancer. However, the precise underlying mechanisms remain unclear. This study aimed to investigate the molecular mechanisms of RA in HepG2 cells. RA anti-tumor activity was assessed using 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays, and Hoechst 33258 staining. Apoptosis and the cell cycle distribution were evaluated by flow cytometry. A proteomics approach was used to identify differentially expressed proteins following RA treatment in HepG2 cells, and quantitative reverse transcription–quantitative polymerase chain reaction was used to validate the results. Bioinformatics analysis was also implemented to further understand the identified proteins, and western blotting was used to analyze the associated proteins. Our results suggested that RA treatment significantly inhibits the viability of HepG2 cells. The MTT and LDH assays indicated dose-dependent decreases in cell proliferation following RA treatment. Hoechst 33258 staining and flow cytometry analysis showed that RA exhibits an apoptosis-inducing effect and induces cell cycle arrest in G1. The proteomics analysis successfully identified 16 differentially expressed proteins. Bioinformatics analysis indicated that the identified proteins participated in several biological processes and exhibited various molecular functions, mainly related to inactivation of the glycolytic pathway. Further western blotting analysis showed that RA could downregulate the expression of glucose transporter-1 and hexokinase-2, leading to the suppression of glucose consumption and generation of lactate and ATP. Taken together, our study found that RA exhibits significant cytotoxic effects by inhibiting cell proliferation and inducing apoptosis and cell cycle arrest, possibly by blocking the glycolytic pathway in human HepG2 cells.