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Background: Some studies of Saccharomyces cerevisiae and mammals have shown that RIO protein kinases (RIOKs) are involved in ribosome biogenesis, cell cycle progression and development. However, there is a paucity of information on their functions in parasitic nematodes. We aimed to investigate the function of RIOK-1 encoding gene from Strongyloides stercoralis, a nematode parasitizing humans and dogs. Methodology/Principal Findings: The RIOK-1 protein-encoding gene Ss-riok-1 was characterized from S. stercoralis. The full-length cDNA, gDNA and putative promoter region of Ss-riok-1 were isolated and sequenced. The cDNA comprises 1,828 bp, including a 377 bp 5′-UTR, a 17 bp 3′-UTR and a 1,434 bp ORF encoding a protein of 477 amino acids containing a RIOK-1 signature motif. The genomic sequence of the Ss-riok-1 coding region is 1,636 bp in length and has three exons and two introns. The putative promoter region comprises 4,280 bp and contains conserved promoter elements, including four CAAT boxes, 12 GATA boxes, eight E-boxes (CANNTG) and 38 TATA boxes. The Ss-riok-1 gene is transcribed throughout all developmental stages with the highest transcript abundance in the infective third-stage larva (iL3). Recombinant Ss-RIOK-1 is an active kinase, capable of both phosphorylation and auto-phosphorylation. Patterns of transcriptional reporter expression in transgenic S. stercoralis larvae indicated that Ss-RIOK-1 is expressed in neurons of the head, body and tail as well as in pharynx and hypodermis. Conclusions/Significance: The characterization of the molecular and the temporal and spatial expression patterns of the encoding gene provide first clues as to functions of RIOKs in the biological processes of parasitic nematodes. Author Summary: Parasitic nematodes cause serious global health problems and enormous economic losses. Control of these parasites is difficult due to their complicated life cycle and the lack of knowledge of their developmental biology at the molecular level. Protein kinases are key molecules regulating a range of biological processes of organisms. The atypical protein kinase RIOK-1 was reported to be indispensable in yeast, as well as in free-living nematode Caenorhabditis elegans, but little is known about its function in parasitic nematodes. In the present study, we investigate the RIOK-1 encoding gene (Ss-riok-1) and its predicted protein Ss-RIOK-1 from parasitic nematode Strongyloides stercoralis which causes canine and human diseases. We found that Ss-RIOK-1 has high sequence identities (50–65%) to its homologues from both vertebrates and invertebrates. It also has abilities of phosphorylation and auto-phosphorylation in vitro. Ss-riok-1 transcript is present in all stages of S. stercoralis with more abundance in the parasitic stages than in the free-living stages, along with the gene expression in neuron system of post free-living L1 and body muscle of iL3, indicating that it plays important role in the development and infection of S. stercoralis. The findings have important implications for understanding the function of RIOK-1 in the development of parasitic nematodes. [ABSTRACT FROM AUTHOR]