Gut microbiome critically contributes to host health status. Thus investigating the relationship between gut microbiome and toxic chemicals is a hot topic in toxicology research. Exposure to malachite green (MG) has been linked to various health disorders. Thus, exploring the gut microbiota changes in response to MG would provide a new perspective on the toxicity effects of this chemical substance. MG exposure resulted in the significantly lower alpha diversity but higher beta diversity of gut microbiota, and significantly decreased ecosystem stability of gut microbial communities. Gut bacterial networks showed that the interactions within network became more complex and stronger interactions after MG exposure, which could decrease the networks stability. Changed in gut microbiota composition was mainly reflected in the overgrowth of opportunistic bacteria (i.e., Aeromonas and Vibrio) and the depression of fermentative bacteria (i.e., Bacteroides and Paludibacter). MG exposure led to the significant increased gut permeability, which could reduce the host selective pressures on particular bacterial species (such as members in Aeromonas and Vibrio). This result was further supported by the weakened importance of deterministic assembly after MG exposure. All these findings indicated that MG exposed fishes are more susceptible to infection, as evidenced by more abundant potential pathogens, promoted immune activities and increased gut permeability, and further greatly improve our understanding on the toxicity effects of MG.