The bioluminescence of entomopathogenic bacteria of the genus Photorhabdus was considered to be constitutive. However, it has recently been shown that under heat shock conditions, lux-operon is activated in Photorhabdus temperata cells. In the present study, bioluminescence thermoactivation was investigated in two isolated strains, Photorhabdus hainanensis FV2401 and P. temperata FV2402. The luminescence of the more thermotolerant P. hainanensis cells was activated at higher temperatures than that of P. temperata. At the maximum induction temperatures of 37 °C for FV2401 and 34 °C for FV2402, higher bioluminescence activation was observed in FV2401, whereas the increase in lux-genes mRNA levels was approximately the same in both strains. In P. hainanensis, elevated temperatures also affected the expression of the hexA, madB, and csrB genes, which are indirectly involved in luminescence in Photorhabdus. The heat-activated lux-operon promoter in P. hainanensis and P. temperata was determined. This is a σ⁷⁰-dependent promoter that differs in its distance from the luxC start codon. When promoter variants with or without the 5′-UTR mRNA were transferred to the heterologous Escherichia coli system, heat activation of luminescence was retained in constructs containing only the promoter and was independent of σ³² and σ^E, whereas constructs with the 5′-UTR exhibited repression of luminescence following thermoactivation. The obtained data on the temperature-induced promoter of the lux-operon can broaden understanding of the ecological and biological functions of Photorhabdus bioluminescence under heat shock conditions and provide a basis for the development of novel biosensors.