Smoking status and duration are associated with taxonomic and functional differences in the oral microbiome

Smoking is a major modifiable risk factor for human health and has been linked to alterations in the oral microbiome. However, how oral microbial composition and functional potential differ across smoking duration remains insufficiently characterized. This study aimed to evaluate the associations of smoking status and duration with the oral microbiome in a large adult population. This cross-sectional study included 2,390 adults from a population-based cohort in China. Oral swab samples were analyzed using 16S rRNA gene sequencing, and a subset of 856 participants underwent shotgun metagenomic sequencing. Microbial diversity, taxonomic composition, and functional profiles were characterized. Community-level associations were assessed using PERMANOVA, while associations between smoking and microbial features were evaluated using MaAsLin2 with adjustment for potential confounders. Generalized additive models were applied to assess nonlinear associations between smoking duration and microbial features. Functional pathways were profiled using HUMAnN (version 3.9), and network analysis was used to explore species–pathway associations. Smoking was significantly associated with oral microbiome variation and explained more microbial compositional variation than other behavioral factors. Specifically, smoking status explained 1.89% of community variation ( p = 0.001), while smoking duration also showed a significant association with community structure (R 2 = 0.0101, p = 0.001). Smokers exhibited distinct microbial profiles characterized by decreased abundance of health-associated commensals such as Neisseria, Haemophilus , and Lautropia , and increased abundance of anaerobic and periodontitis-associated genera including Megasphaera , Veillonella , Actinomyces , and Granulicatella. Alpha diversity showed a nonlinear relationship with smoking duration, increasing at early and intermediate exposure levels and stabilizing or slightly decreasing at higher exposure. Shotgun metagenomic profiling identified smoking-associated differences in pathways related to central carbon metabolism, biosynthesis, and nitrate reduction. Correlation network analysis further identified coordinated statistical associations among smoking status, microbial species, and metagenomic pathways, with several Neisseria species occupying a central position in the major species–pathway module. Smoking is associated with alterations in the oral microbiome at both the compositional and functional levels. These patterns varied across smoking duration, with longer duration generally associated with more pronounced taxonomic and functional differences. Overall, these findings provide new evidence for smoking-related oral microbiome dysbiosis.