Abstract: Livestock manure has been regarded as an important reservoir of antibiotics and antibiotic resistance genes (ARGs). However, most livestock manure is stacked directly in the farm, which causes a potential threat to the surrounding soil and groundwater safety. In order to study the effect of long-term livestock manure stacking on the vertical distribution of ARGs and bacterial communities, 0−100 cm soil samples were collected from pig and chicken manure stacking sites, respectively. Real-time quantitative PCR results showed that pig and chicken manure stacking increased the detection ratio and abundance of tetracycline resistance genes (tetC, tetG, tetL, tetW) and sulfonamide resistance genes (sulI, sulII), and an integrase gene (intI1) in soil samples. This demonstrated that livestock manure stacking could lead to the contamination of ARGs in the surrounding soil. According to the cluster analysis, the abundance of ARGs and intI1 gene were decreased with increasing soil depth and mainly concentrated in the 0−30 cm soil layer, which posed a risk of migration of ARGs into the deep soil. In addition, intI1 gene had a significant and positive correlation with ARGs abundance, indicating that intI1 gene may play an important role in disseminating ARGs. Furthermore, the high-throughput sequencing results showed that both pig and chicken manure stacking significantly reduced and changed the diversity of bacterial communities in the 0−10 cm and 10−30 cm soil layers, compared with the control soil. The difference of bacterial community structure between livestock manure stacking site soil and control soil was higher in 0−30 cm than in deep soil. What’s more, both soil chemical properties and bacterial community affected the vertical distribution of ARGs in soil, with the shift of bacterial community structure representing the major driver shaping the ARGs distribution based on variation partitioning analysis. Taken together, our results provide insight into the control of ARGs pollution in livestock manure stacking stie soil around the farms.