Though being able to encode various kinds of bioactive peptides, small open reading frames (sORFs) are poorly annotated in many genomic data. The present study was conducted to evaluate the potential of sORFs in encoding antimicrobial peptides (AMPs) in the basal chordate modelCiona intestinalis. About 4.8 mgenomic sequence was first retrieved for sORFs mining by the program sORFfinder, then the sORFs were translated into amino acid sequences for AMP prediction via CAMP server, and thereafter, ten putative AMPs were selected for expression and antimicrobial activity validation. In total, over 180 peptides deduced from the sORFs were predicted to be AMPs. Among the ten tested peptides, six were found to have significant expressed sequence tag matches, providing strong evidence for gene expression; five were proved to be active against the bacterial strains. These results indicate that many sORFs inC. intestinalisgenome contain AMP information. This work can serve as an important initial step to investigate the role of sORFs in the innate defense ofC. intestinalis. Copyright © 2013 European Peptide Society and John Wiley&Son