Background <p>Betel nut consumption is closely related to oral cancer. This study aimed to explore the crucial target and role of betel nut in oral cancer.</p> Methods <p>We used RNA sequencing and protein-protein interaction networks to screen the key target of betel nut in oral cancer. Enrichment analysis was used to explore the role of betel nut. Immunohistochemical analysis was used to identify the expression trend of the key target, and survival analysis was used to explore the prognostic value of the key target. The animal model of oral cancer was used to verify the key target of betel nut.</p> Results <p>Firstly, 1108 genes were differentially expressed in oral cancer under the influence of betel nut, which may be involved in “metabolic pathways.” Moreover, TP53 was the key target of betel nut and was upregulated, resulting in poor prognosis in patients with oral cancer. Finally, the differential expression and role of TP53 in oral cancer with betel nut were validated by human and animal samples.</p> Conclusions <p>In conclusion, we found that TP53 upregulation may be the crucial biological phenomenon in the oral cancer with betel nut, indicating that TP53 may be the key target of betel nut in oral cancer.</p>