Vanadium (V) and microplastics in soils draw increasing attention considering their significant threats to ecosystems. However, little is known about the vertical co-distribution of V and microplastics in soil profile and their combined effects on microbial community dynamics and assembly. This study investigated the spatial distribution of V and microplastics in the soils at a V smelting site and the associated microbial community characteristics along the vertical gradient. Both V and microplastics were found in the 50 cm soil profile with average concentrations of 203.5 ± 314.4 mg/kg and 165.1 ± 124.8 item/kg, respectively. Topsoil (0-20 cm) and subsoil (20-50 cm) displayed distinct microbial community compositions. Metal-tolerant (e.g., Spirochaeta, Rubellimicrobium) and organic-degrading (e.g., Bradyrhizobium, Pseudolabrys) taxa as biomarkers were more abundant in the topsoil layer. V and microplastics directly affected the microbial structure in the topsoil and had indirect influences in the subsoil, with direct impacts from organic matter. In topsoil, deterministic processes were more prevalent for community assembly, whereas stochastic processes governed the subsoil. The interspecific relationship was closer in topsoil with greater network complexity and higher modularity. These findings promote the understanding of distinct heterogeneity of microbial communities jointly driven by V and microplastics in soil environment.