Rice bran is an economical, under-utilized co-product of rough rice milling. The objective of this study was to produce rice-bran peptides and investigate for anti-cancer activity. Protein hydrolysates were prepared by treating heat stabilized defatted rice-bran with food grade Alcalase enzyme, followed by treatment with simulated gastric and intestinal juices to obtain resistant peptides. Resistant peptides were fractionated into >50, 10-50, 5-10, and <5 kDa sizes, freeze dried, and evaluated for inhibitory and cytotoxicity activities on human colon (HCT-116) and breast (HTB-26) cancer cell lines. The results showed that <5 kDa fraction of rice-bran is a potent anti-cancer agent. The cytotoxicity of the fraction to both cancer cell types was more pronounced after the treatment with 500 µg/mL. The IC₅₀ of the peptide fraction was approximately 750 µg/mL. These results indicate that the <5 kDa peptide fraction separated from rice bran protein hydrolysate has a potent anti-tumor activity for colon cancer cells. The peptide fractions that demonstrate anti-cancer activities have the potential for use as functional food ingredients for health benefits.