Two novel Gram-stain negative facultative anaerobic, motile, rod-shaped bacterial strains IG-V01T and IG-V01b were isolated from the gut of red flour beetles, Tribolium castaneum. The 16S rRNA gene sequences of strains IG-V01T and IG-V01b were found to have their highest sequence similarity (96.5 % and 96.4 %) with Serratia nematodiphila DZ0503SBS1T (Enterobacteriaceae family) respectively. Strains IG-V01T and IG-V01b share 100 % 16S rRNA gene sequence similarity and exhibit very similar phenotypic characteristics. In addition, they show 89.7 % genomic relatedness (DNA–DNA hybridisation). Major fatty acids were identified to be C16:0 (38.3 %), C17:0 cyclo (19.5–20 %) and C14:0 (11.2–11.3 %). Cells contain phosphatidylethanolamine and diphosphatidylglycerol as predominant polar lipids. Genomic DNA G+C content (mol%) was determined to be 51.5–51.7. A polyphasic approach employing the study of morphological, physiological, chemotaxonomic, genomic and phylogenetic analysis revealed that the two newly isolated strains cannot be placed in any of the existing genera of the family Enterobacteriaceae. Therefore, it is proposed that strains IG-V01T and IG-V01b belong to a novel genus within the family Enterobacteriaceae, and represent a new species Enterobacillus tribolii gen. nov., sp. nov., with the type strain =IG-V01T = KCTC 42159T = MCC 2532T.