In order to explore the role of rhamnose-binding lectin (RBL) in the non-specific cell defense of bony fishes against pathogen infection. The head kidney monocytes/macrophages from Nile tilapia (Oreochromis niloticus) were isolated to conduct a bacterial stimulation experiment in vitro. The expression of Onrbl-1 was significantly up-regulated following challenges with two important pathogens, Streptococcus agalactiae and Aeromonas hydrophila. Meanwhile, OnRBL-1 recombinant protein could participate in the regulation of the expression of pathogen-induced cell inflammatory factors by quantitative real-time PCR (qRT-PCR), including significantly inhibiting the expression of il-6, il-8 and tnf-α, and promoting the expression of il-10 and tgf-β. In addition, the results showed that OnRBL-1 could promote the phagocytosis, enhance the level of respiratory burst and the release of reactive oxygen species in monocytes/macrophages through flow cytometry analysis. Taken together, the results indicated that OnRBL-1 plays an important regulatory role in the non-specific cellular defense of monocytes/macrophages from O. niloticus. This study provides a reference for exploring the function of RBL-1 in host defense against pathogen infection, which helps to explain the action mechanism of bony fish RBL in the defense against pathogen infection and improve the basic theoretical system of its participation in immune response, with great significance in science.