<p>Cold seep is a distinctive deep-sea environment mainly formed by methane-rich fluids leaking on the seafloor, gaps remain regarding the influence of seepage on microorganisms inhabiting water column across vertical and horizontal dimensions. Site F cold seep, located at 1,120&#xa0;m depth on the northern South China Sea (SCS) slope, is one of the most active cold seeps in SCS. We performed 16S rRNA gene and metagenomic sequencing on samples collected by Niskin bottles mounted on Conductivity-Temperature-Depth profiler and Remote Operated Vehicle to analyze the structure and metabolic potentials of microbial communities throughout the water column at Site F. Microbial abundance generally decreased with depth at all sampling spots and was higher at sites adjacent to the seepage compared to those farther away, indicating a potential vertical and horizonal influence of methane seepage on water microbial community. High microbial abundance at deeper depths may attribute to a higher proportion of <i>Gammaproteobacteria</i>, comprised mainly of <i>Alcanivoracaceae</i>, <i>Alteromonadaceae</i>, <i>Marinobacteraceae</i>, methylotrophs represented by <i>Methylophagaceae</i> and <i>Methylococcales</i> (mainly <i>Methylomonadaceae</i>), and sulfur-oxidizing bacteria represented by SUP05 and <i>Ectothiorhodospiraceae</i>. Consistently, the aerobic methane oxidation gene <i>pmoA</i> was more prevalent in the deeper water and was found in four bacterial classes in addition to <i>Gammaproteobacteria</i>. Sulfur-oxidizing genes also exhibited higher abundances at depths and were primarily affiliated with <i>Rhodobacteraceae</i>. These microbes likely play important roles in aerobic oxidation of methane and sulfur, contributing to methane depletion during upward diffusion. By integrating sampling across vertical and horizontal dimensions, we demonstrate that seepage shapes the microbial community and biogeochemical functions in the water column at Site F.</p>