Bacterial virulence is an integrative process that may involve quorum sensing. In this work, we compared by global expression profiling the wild-type entomopathogenic Photorhabdus luminescens subsp. laumondii TT01 to a luxS-deficient mutant unable to synthesize the type 2 quorum-sensing inducer AI-2. AI-2 was shown to regulate more than 300 targets involved in most compartments and metabolic pathways of the cell. AI-2 is located high in the hierarchy, as it controls the expression of several transcriptional regulators. The regulatory effect of AI-2 appeared to be dose dependent. The luxS-deficient strain exhibited decreased biofilm formation and increased type IV/V pilus-dependent twitching motility. AI-2 activated its own synthesis and transport. It also modulated bioluminescence by regulating the synthesis of spermidine. AI-2 was further shown to increase oxidative stress resistance, which is necessary to overcome part of the innate immune response of the host insect involving reactive oxygen species. Finally, we showed that the luxS-deficient strain had attenuated virulence against the lepidopteran Spodoptera littoralis. We concluded that AI-2 is involved mainly in early steps of insect invasion in P. luminescens.