Extensive studies of the well-known legume and rhizobium symbiosis model system suggest that the purine metabolic pathway plays a key role in microbe-plant interactions, although the exact mechanism is unknown. Here, we report the impact of a key purine metabolic gene, purL, on the symbiotic interaction between the bacterium Photorhabdus temperata and its nematode partner Heterorhabditis bacteriophora. Real-time PCR assays showed that the purL gene was upregulated in P. temperata in the nematode infective juvenile compared with artificial media. Mutation of the purL gene by in-frame deletion dramatically decreased the capacity of the bacterium to persist in infective juveniles and its ability to form biofilm in vitro. It was further demonstrated that purL gene expression was positively related to bacterial biofilm formation and the symbiotic persistence of the bacterium in nematode infective juveniles. A ΔpurL mutant lost the ability to support infective juvenile formation in the media which weakly supported biofilm formation, suggesting that a critical level of biofilm formation is required by the bacteria to support infective juvenile formation and thus establish their partnership. In addition, the defects in both biofilm formation and symbiotic ability due to the disruption of the purL gene could be partially restored by the addition of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an intermediate of the purine biosynthesis pathway. Overall, these data indicate that the purine metabolic pathway is important in microbe-animal symbioses, and that it may influence symbiotic interactions at the level of biofilm formation.