Abstract: 50%–98% of crude oil is made up of hydrocarbons, and 20%–45% of them are cycloalkanes. The content and hazards of cycloalkanes are very high and have obvious toxic effects on aquatic animals and humans. However, there is limited research work on microbial degradation of cycloalkanes home and abroad. Between 140°E and 35°N, where the North Pacific Kuroshio separated from the Japanese shore, lies the Kuroshio Extension (KE), a region with extremely complicated hydrodynamic processes and a very diverse microbial population. In this study, we used methylcyclohexane (MCH) as the only carbon source and energy source to enrichment culture and subculture seawater in the surface, middle, and bathypelagic layers of KE, and analyzed the microbial diversity and community composition of cycloalkane degrading bacteria in KE by 16S rRNA high-throughput sequencing technology. The addition of MCH considerably decreased the richness and diversity of microbial communities, according to the results of α diversity analysis (P˂0.01). The addition of MCH caused a significant change in the structure of the microbial community and the formation of significantly separate microbial communities compared to the natural microbial communities (P˂0.01) when the community compositions between treatment groups were found different using PLS-DA analysis. In the middle and bathypelagic waters of KE, MCH-degrading bacteria were found, primarily belonging to the genera Cobetia, Pseudoalteromona, Alteromonas, Paraglaciecola, Salinicola, Halomonas, and the putative new genus C1-B045, according to the study of community composition. This study adds to our understanding of the variety of cycloalkane-degrading microorganisms present in the KE and provides new microbial resources required for bioremediation related to oil pollution in marine environments, serving as a foundation for the later in-depth exploitation of microbial resources in the KE.