Cigarette smoking is posited as a potential factor in disrupting the balance of the human gut microbiota. However, existing studies with limited sample size have yielded inconclusive results. Here, we assessed the association between cigarette smoking and gut microbial profile among Chinese males from four independent studies (N total = 3,308). Both 16S rRNA and shotgun metagenomic sequencing methods were employed, covering 206 genera and 237 species. Microbial diversity was compared among non-smokers, current smokers, and former smokers. Actinomyces[g], Atopobium[g], Haemophilus[g], Turicibacter[g], and Lachnospira[g] were found to be associated with smoking status (current smokers v.s. non-smokers). Metagenomic data provided a higher resolution at the species level, particularly for the Actinomyces[g] branch. Additionally, serum Trans-3-Hydroxycotinine was found to have a potential role in connecting smoking and Actinomyces[g]. Furthermore, we revealed putative mediation roles of gut microbiome in the associations between smoking and common diseases including cholecystitis and type 2 diabetes. In conclusion, we characterized the gut microbiota profile in male smokers and further revealed their potential involvement in mediating the impact of smoking on health outcomes. These findings advance our understanding of the intricate associations between cigarette smoking and the gut microbiome.