The Colorado Potato Beetle (CPB), Leptinotarsa decemlineata (Say) (Col.: Chrysomelidae), is a pre-eminent pest on solanaceous crops worldwide, and much research has focused on the different components of its variable population dynamics. L. decemlineata has developed resistance to insecticides used for its control. Consequently, alternative methods are needed urgently for the control of this pest. In this study, in order to find a more effective and safer biological control agent against L. decemlineata, we isolated a new bacterial isolate (Ld4) from L. decemlineata, and tested it for insecticidal effects on L. decemlineata larvae. Also, the insecticidal effect of Ld4 was compared with Ld3, which was isolated from L. decemlineata. According to the morphological, physiological, biochemical tests, API20E and API50CH panel test systems, and 16S rRNA sequence homologies, this isolate was identified as Pseudomonas putida. The highest insecticidal effect determined on L. decemlineata within 5 days was 100%, and this activity was exhibited by Ld4 isolate. Our results indicate that Ld4 isolate may be valuable as a biological control agent for the biological control of L. decemlineata.