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[Objectives] This study focused on interaction mechanism between H. rugaoensis/S. nematophila R187 complex and humoral immune system in Drosophila melanogaster infected by the nematode/bacteria complex. [Methods] Three methods, nematode with symbiotic bacteria strain HI87 infecting flies, needle-pricking the fly with the symbiotic bacterial strain R187 and feeding flies with symbiotic bacterium, were conducted to tranfer the bacterial strain H187 into hemocoel or intestine of flies. Lethal rate of flies and colonies statistic of symbiotic bacterium combined with Q-PCR detection of expression of the two antimicrobial peptides Diptericin and Drosomycin regulated respectively by Imd and Toll pathway were analyzed to recover the interaction between H. rugaoensis-S. nematodiphila R187 and the humoral immune system in D. melanogaster. The fly stocks used in this experiment were the wild type, FADD mutant( Imd) and Dif mutant (Toll). [Results] Infecting of H. rugaoensis with its symbiotic bacterial strain R187 or needle-pricking with the bacterial strain on flies could activate both the two humoral immune responses, Toll and Imd pathway. Results of fluorescence quantitative PGR detection showed that expression of Diptericin and Drosomycin in the two infecting methods had the same trend that up-regulated firstly and then gradually down-regulated. In the needle-pricking infection with symbiotic bacterium R187, the relative expression level of Drosomycin in FADD mutants was in the highest(100%)in 6 h, and those of Diptericin in Dif mutant and wild type were in the highest (90%) in 12 h. Only very weak expression of Diptericin and Drosomycin were detected in intestine infection of S. nematodiphila R187 on flies. All the three infecting methods could result in 100% lethal of flies. Lethal process of Dif mutant and wild type was similar, slowly than that of FADD mutants. Needle-pricking method had the highest infect efficency and caused 100% death of flies in 20 h. [ Conclusions] The rhahditid entomopathogenic nematode H. rugaoensis and its symbiotic bacterial strain S. nematophila R187 infected hemocoel of adult D. melanogaster together or separately could activate humoral immune responses of both Toll and Imd pathway in flies. Imd was the main immune response pathway for fly when faced with the infection of S. nematophila R187. There might be some immune escape mechanism to help S. nematodiphila R187 to infect flies successfully in the process of intestinal infection. [ABSTRACT FROM AUTHOR]