Globally, Nicotiana tabacum (common tobacco) and Nicotiana rustica (aztec tobacco) are the most prevalently utilized species among the 70 identified species of Nicotiana for various forms of consumption such as smoking, chewing, and snuffing. Prior research has established a link between tobacco use and an increased risk of cancer, alongside neurological and cardiac complications. This study embarked on exploring the complex interaction between miRNAs derived from tobacco and the regulatory mechanisms they may exert on human transcripts within the 3’ untranslated regions (UTRs), employing a combination of computational and experimental genomic strategies. Marking a pioneering venture into cross-kingdom analysis, this research predicted tobacco-specific miRNAs utilizing express sequence tags (EST), genome-wide analyses, and deep sequencing techniques. Through rigorous bioinformatics tools, plant targets of these miRNAs were pinpointed. An extensive examination was conducted on 164 mature miRNAs, along with 38 miRNAs identified from EST datasets and 21 from genomic data, to explore their crosskingdom regulatory potential. Remarkably, our analysis uncovered that certain tobaccoderived miRNAs, including miR156, miR403, miR7997, miR8011, and miR8036, target genes known for their tumor-suppressing functions, implicating them in carcinogenic pathways. This research identified an exhaustive list of miRNAs through small RNA sequencing of N. rustica, unveiling 842 novel miRNAs with potential targets in critical cancer pathways such as AMPK and WNT. This groundbreaking study establishes the first documented evidence of the interaction between N. tabacum-derived miRNAs and human genes, setting the stage for targeted future investigations into the specific cancer-related pathways influenced by tobacco-derived miRNAs