Urea is one of the dominant organic nitrogenous compounds in the oligotrophic oceans. Compared to the knowledge of nitrogen transformation of nitrogen fixation, ammonia oxidization, nitrate and nitrite reduction mediated by sponge-associated microbes, our knowledge of urea utilization in sponges and the phylogenetic diversity of sponge-associated microbes with urea utilization potential is very limited. In this study, Marinobacter litoralis isolated from the marine sponge Xestospongia testudinaria and the slurry of X. testudinaria were found to have urease activity. Subsequently, phylogenetically diverse bacterial ureC genes were detected in the total genomic DNA and RNA of sponge X. testudinaria, i.e., 19 operative taxonomic units (OTUs) in genomic DNA library and 8 OTUs in cDNA library at 90% stringency. Particularly, 6 OTUs were common to both the genomic DNA library and the cDNA library, which suggested that some ureC genes were expressed in this sponge. BLAST and phylogenetic analysis showed that most of the ureC sequences were similar with the urease alpha subunit of members from Proteobacteria, which were the predominant component in sponge X. testudinaria, and the remaining ureC sequences were related to those from Magnetococcus, Cyanobacteria, and Actinobacteria. This study is the first assessment of the role of sponge bacterial symbionts in the regenerated utilization of urea by the detection of transcriptional activity of ureC gene, as well as the phylogenetic diversity of ureC gene of sponge bacterial symbionts. The results suggested the urea utilization by bacterial symbionts in marine sponge X. testudinaria, extending our understanding of nitrogen cycling mediated by sponge-associated microbiota.