Simple SummaryPlutella xylostellais a major pest of Cruciferae vegetables all over the world. Gut bacteria play an important role in the life activities ofP. xylostella, but so far, little is known about the source and transmission of gut bacteria ofP. xylostella. Therefore, we used the traditional microbial culture method to show that there is a potential correlation between the gut bacteria ofP. xylostellaand food bacteria, andP. xylostellagut bacteria exhibit vertical and horizontal transmission through eggs. Our research results will contribute to biological pest control based on gut bacteria.AbstractPlutella xylostella(L.), commonly known as the diamondback moth, is currently a major worldwide pest. Gut bacteria play an important role in the physiology and insecticide resistance ofP. xylostella, but little is known about the sources and transmission routes of its gut bacteria. In this study, traditional microbial culture methods were used to analyze the sources and transmission modes of gut bacteria inP. xylostella, which could help develop pest control strategies based on gut bacteria. The main findings are as follows: gut bacterial diversity was significantly higher inP. xylostella-fed radish sprouts than those fed an artificial diet, indicating a potential association between gut bacteria and food bacteria. In addition, sequence analysis revealed the isolation ofEnterobactersp.,Pantoeasp.,Cedeceasp., andPseudomonassp. from both radish sprouts andP. xylostella. Importantly,Enterobactersp. was found in all tested samples (radish sprouts, gut ofP. xylostella, ovaries, and eggs), suggesting that bacteria acquired from food could be transferred from the gut to the ovaries and eggs. This was confirmed through experiments, which also showed that eggs could carry bacteria and transmit them to the gut, indicating vertical transmission of gut bacteria via eggs. Furthermore, the 3rd instar larvae ofP. xylostellawith and without gut bacteria were mixed and raised until the 4th instar. Then, we found that all the gut of the 4th instar larvae carried the same bacteria, indicating that the gut bacteria ofP. xylostellacan be horizontally transmitted through social behavior. This study lays a foundation for further exploration of the sources, transmission, and coevolution of the host of gut bacteria inP. xylostella, and provides new insights into pest control strategies based on the source and transmission of gut bacteria.