Ankaferd hemostat (ABS), a traditional herbal extract, is a hemostatic agent used for wound healing and bleeding treatment. A standardized form of plants contains many biomolecules. In recent years, previous studies have demonstrated the antineoplastic effect of ABS. In the present work, we focused on the mechanism of its antineoplastic effect over Caco-2 colon cancer cells. The LC/MS-based proteomics method was used to understand the effect of ABS at the protein level. The results were evaluated with gene ontology, protein interaction, and pathway analysis. As shown by our results, ABS altered glucose, fatty acids, and protein metabolism. Moreover, ABS affects the cell cycle machinery. Moreover, we found that ABS induced critical cancer target and suppressor proteins such as carboxyl-terminal hydrolase 1, 60S ribosomal protein L5, Tumor protein D52-like2, karyopherin alpha 2, and protein deglycase DJ-1. In conclusion, the proteomics results indicated that ABS affects various cancer targets and suppressor proteins. Moreover ABS has systematical effect on cell metabolism and cell cycle in Caco-2 cells, suggesting that it could be used as an antineoplastic agent.