Phenylacetylglutamine (PAGln), a gut microbiota (GM)-derived metabolite, is associated with cardiovascular disease. Studies have shown that disordered GM participated in the progression of atrial fibrillation (AF), but the relationship between PAGln and AF is unclear. This study investigated the characteristics of PAGln in AF patients and its impact on atrial myocytes. Based on our previous metagenomic data, the relative abundance of porA, a critical bacterial enzyme for PAGln synthesis, exhibited an increased tendency in AF. In an independent cohort consisting of 42 controls without AF and 92 AF patients, plasma PAGln levels were higher in AF patients than in controls (p < 0.001) by immunoassay. Notably, PAGln exerted a predictive potential of AF with an AUC of 0.774 (p < 0.001), and a predictive model constructed based on the PAGln and Taiwan AF score further improved the predictive potential. Furthermore, a positive correlation was determined between PAGln and LA diameter. Subsequently, the effect of PAGln intervention was examined on HL-1 cells in vitro, revealing that PAGln increased apoptosis, reactive oxygen species (ROS) production, CaMKII and RyR2 activation and decreased cell viability. In conclusion, increased PAGln was associated with AF, and PAGln might contribute to the AF pathogenesis by promoting oxidative stress and apoptosis in atrial myocytes.