Helicase-like proteins play a crucial role in nucleic acid- and chromatin-mediated reactions. In this study, we identified 134 helicase-like proteins in the nematode Caenorhabditis elegans and classified the proteins into 10 known subfamilies and a group of orphan genes on the basis of sequence similarity. We characterized loss-of-function phenotypes in RNA interference (RNAi)-treated animals for helicase family members, using the RNAi feeding method, and found several previously unreported phenotypes. Fifty-one (39.5%) of 129 genes tested showed development- or growth-defect phenotypes, and many of these genes were putative nematode homologs of essential genes in a unicellular eukaryote, budding yeast, suggesting conservation of these essential proteins in both species. Comparative analyses between these species identified evolutionarily diverged nematode proteins as well as conserved family members. Chromosome mapping of the nematode genes revealed 10 pairs of putative duplicated genes and clusters of C. elegans-specific SNF2-like genes and Helitrons. Analyses of transcriptional profile data revealed a predominantly oogenesis- and germline-enriched expression of many helicase-like genes. Finally, we identified the D2005.5(drh-3) gene in an RNAi-based screen for genes involved in resistance to X-ray irradiation. Analysis of DRH-3 will clarify the potentially novel mechanism by which it protects against X-ray-induced damage in C. elegans.