Completely switching from cigarette smoking to oral tobacco-derived nicotine products, like nicotine pouches, presents a harm reduction opportunity for adult smokers who are unable or unwilling to quit using tobacco. The objective of this study was to compare subgingival microbiome profiles in adult smokers who switched from cigarette smoking (CS) to using on!^® nicotine pouches (NPs) in a single-center, randomized, open-label, parallel-group study. Adult smokers with moderate gingivitis were randomly assigned into NP (n=88) and CS (n=61) groups at baseline. Subgingival plaque was collected by curettes from two separate sites with the highest gingival inflammation determined by modified gingival index and bleeding at baseline. Sampling was repeated at 12- and 24-weeks from the same sites. The V1-V3 regions of 16S rDNA were sequenced using the Illumina platform. Alpha (Shannon and Simpson indices) and beta diversities, differential abundance with bias control, and LefSe analyses were determined. In NP users, no statistically significant changes were observed in alpha and beta diversities at both 12- and 24-weeks compared to baseline. However, using LefSe analysis, health-associated species, e.g., Rothia aeria, Streptococcus cristatus, Kingella oralis, species of Neisseria and Haemophilus, became more predominant while relative abundances of putative pathogens, e.g., Fusobacterium, Saccharibacteria TM7 and Prevotella spp. declined at both time points. In the continued smoking group, no statistically significant changes were observed in alpha or beta diversities at 12- or 24-weeks compared to baseline. In these smokers, using differential abundance analysis, one Capnocytophaga specy was statistically higher (p<0.05) at 24 weeks compared to baseline while some, albeit low abundance, differences using LefSe were observed over time.