The gut microbiota plays a key role in physiological processes of insects including nutritional metabolism, development, immunity and detoxification. Environmental stressors such as herbicides, used to optimize and improve crop yields, may interfere with the mutualistic relationships causing negative consequences for the host health. This study assessed the effect of a pendimethalin-based herbicide on the gut microbial community of the carabid beetle Pterostichus melas italicus Dejean, 1828 (Coleoptera, Carabidae), a generalist predator in the soil food web of agroecosystems. The exposure effect was tested in vivo by using a recommended field rate and evaluating the variability of responses in 21 days, corresponding to the half-life of the pendimethalin. The 16S rRNA sequencing data showed that the gut lumen was dominated by Proteobacteria, Firmicutes, Fusobacteria, Tenericutes and Bacteroidetes. The herbicide exposure interfered with the bacterial community richness and diversity associated with the gut from 2 days after the treatment. The differential abundance analyses highlighted a shift involving families such as Lactobacillaceae, Streptomycetaceae, Neisseriaceae and Ruminococcaceae and genera belonging to Enterobacteriaceae. An increase of bacterial species such as Enterobacter sp., Pseudomonas sp., Pantoea sp and Paracoccus sp. involved in the herbicide degradation was also recorded after 21 days of exposure. Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) analysis indicated that the exposure have effects on the most predicted functional categories of gut microbiota related to metabolic function including carbohydrate, amino acid and lipid metabolism of P.melas. These results demonstrate that pendimethalin can impact microbial communities associated with generalist predators inhabiting croplands leading to severe implications for the species ecological role. Understanding the effects of herbicides such as pendimethalin on ground beetles may help to protect beneficial soil insects that have a crucial role in the ecosystem services.