ABSTRACT The antioxidant and antiproliferative activities of a methanol extract from hot pepper (Capsicum annuum L.) seeds (pepper seed extract [PSE]) were evaluated. The polyphenolic content of the PSE was 10.9 ± 0.0 mg gallic acid equivalents/g residue, and the capsaicin and dihydrocapsaicin contents were 22.7 and 6.5 mg/100 g of residue, respectively. The 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) and 1,1-diphenyl-2-picrylhydrazyl radical scavenging activities of hot pepper seeds were 38.6 ± 0.7 and 20.8 ± 2.8 Trolox equivalent antioxidant capacity (mg/g residue), respectively. Pretreatment with PSE (500 µg/mL) significantly reduced (P < 0.05) reactive oxygen species generation in HepG2 cells compared with the control; however, PSE at low concentrations (10–200 µg/mL) had no effect on cellular antioxidation. PSE showed strong antiproliferative activity against MCF7 (73.5%), MKN45 (96.8%) and HCT116 (80.0%) tumor cells at a concentration of 500 µg/mL because of an increase in apoptosis. PRACTICAL APPLICATIONS Agricultural and industrial residues are attractive sources of potential natural antioxidants. Pepper seeds are obtained as by-products in the preparation of red pepper powder and constitute 4.14% of pepper by fresh weight. In this study, hot pepper seeds were evaluated as a new source of antioxidant and anticancer agents and as a functional material from agricultural waste. Pepper seed extract showed relatively low antioxidant activity and polyphenolic content but exerted profound antiproliferative effects on tumor cells, even at low concentration.