Urease-producing bacteria (UPB) provide inorganic nitrogen for primary producers by hydrolyzing urea, and play an important role in marine nitrogen cycle. However, there is still an incomplete understanding of UPB and their ecological functions in the cultivation environment of the red macroalgae Gracilariopsis lemaneiformis. This study comprehensively analyzed the diversity of culturable UPB and explored their effects on urea uptake by G. lemaneiformis. A total of 34 isolates belonging to four main bacterial phyla i.e. (Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria) were identified through 16S rRNA sequencing and were screened for UPB by urea agar chromogenic medium assay and ureC gene cloning. Our data revealed that only 8 strains contained urease. All of these UPB exhibited different urease activities, which were determined by the Berthelot reaction colorimetry assay. Additionally, the UPB strain (G13) isolated from G. lemaneiformis with higher urease activity was selected for co-culture with G. lemaneiformis to explore its role in promoting or inhibiting nitrogen uptake by macroalgae. The results showed a significant increase in urea consumption in the culture medium and the total cellular nitrogen in G. lemaneiformis in the UPB-co culture group compared to the sterile group. This suggests that the selected UPB strain positively influences nitrogen uptake by G. lemaneiformis. Similarly, isotopic assays revealed that the δ 15 N content of G. lemaneiformis was significantly higher in the UPB-co culture than in the control group, where δ 15 N-urea was the only nitrogen source in the culture medium. This indicates that the UPB helped G. lemaneiformis to absorb more nitrogen from urea. Moreover, the highest content of δ 15 N was found in G. lemaneiformis with epiphytic bacteria compared to sterilized (i.e. control), showing that epiphytic bacteria, along with UPB, have a compound effect in helping G. lemaneiformis absorb more nitrogen from urea. Taken together, these results provide unique insight into the ecological role of UPB and suggest that urease from macroalgae environment-associated bacteria might be an important player in marine nitrogen cycling.