The insecticidal properties of Bacillus thuringiensis are due mainly to Cry toxins which form a crystalline inclusion during sporulation. However, other proteins participate in the pathogenicity of the bacterium, notably, the Vip3A toxins that are produced from vegetative growth to sporulation. The Vegetative insecticidal protein Vip3A is produced by some Bacillus thuringiensis strains from the mid-log growth phase to sporulation. Although Vip3A is important for the entomopathogenicity of B. thuringiensis, the vip3A gene regulation is unknown. In the B. thuringiensis serovar kurstaki HD1 strain, vip3A is carried by the pBMB299 plasmid, which is absent in the closely related strain B. thuringiensis kurstaki HD73. Using a transcriptional fusion between the vip3A promoter and lacZ, we observed that the HD73 strain is unable to express vip3A. This result suggests that a specific regulator is required for vip3A expression. Assuming that the regulator gene is located on the same plasmid as vip3A, we transferred pBMB299 from the HD1 strain to the HD73 strain. We found that Vip3A was produced in the HD73 strain containing pBMB299, suggesting that the regulator gene is located on this plasmid. Using this heterologous host and promoter-lacZ transcription fusions, we showed that a specific regulator, VipR, is essential to activate vip3A expression at the onset of stationary phase. We demonstrated that vipR transcription is positively autoregulated and the determination of the vipR and vip3A promoters pinpointed a putative VipR target upstream from the Sigma A-specific -10 region of these two promoters. Surprisingly, this conserved sequence was also found upstream of cry1I and cry2 genes. Finally, we showed that vip3A and vipR expression is increased drastically in a Delta spo0A mutant unable to initiate sporulation. In conclusion, we have characterized a novel regulator involved in the entomopathogenic potency of B. thuringiensis through a sporulation-independent pathway. IMPORTANCE The insecticidal properties of Bacillus thuringiensis are due mainly to Cry toxins which form a crystalline inclusion during sporulation. However, other proteins participate in the pathogenicity of the bacterium, notably, the Vip3A toxins that are produced from vegetative growth to sporulation. The VipR regulator that activates vip3A gene expression at the onset of stationary phase is positively autoregulated, and an analysis of the promoter region of the vip3A and vipR genes reveals the presence of a highly conserved DNA sequence. This possible VipR target sequence is also found upstream of the cry2A and cry1I genes, suggesting that Cry toxins can be produced before the bacteria enter sporulation. Such a result could allow us to better understand the role of Cry and Vip3A toxins during the B. thuringiensis infectious cycle in insects, in addition to the primary role of the Cry toxins in the toxemia caused by ingestion of crystals.