The fish gill is the first tissue that is exposed to the external media and undergoes continuous osmotic challenges. Recently, our group published an article entitled “Integrated Omics Approaches Revealed the Osmotic Stress-Responsive Genes and Microbiota in Gill of Marine Medaka” in the journal mSystems (e0004722, 2022), and suggested the possible host-bacterium interaction in the fish gill during osmotic stress. The previous study was performed by the progressive fresh water transfer (i.e., seawater to fresh water transfer via 50% seawater (FW)). Our group hypothesized that osmotic gradient could be a factor that determines the microbiota communities in the gill. The current 16S rRNA metagenomic sequencing study found that the direct transfer (i.e., seawater to fresh water (FWd)) could result in different gill microbiota communities in the same fresh water endpoints. Pseduomonas was the dominant bacteria (more than 55%) in the FWd gill. The Kyoto Encyclopedia of Genes and Genomes and MetaCyc analysis further suggested that the FWd group had enhanced osmosensing pathways, such as the ATP-binding cassette transporters, taurine degradation, and energy-related tricarboxylic acid metabolism compared to the FW group.