Seafloor seeping methane bubbles were successfully sampled on the summit area of the Kuroshima Knoll (depth of ca. 640 m) using the gas-tight sampler WHATS attached to the Shinkai 2000. To evaluate the origin of the bubbles and verify that the dissociation of methane hydrate was actually in progress, the chemical and isotopic composition of the samples were analyzed. The major component of the gas bubbles was methane (C1) with traces of CO2 (67 ± 16 ppmv) and helium (11 ± 1 ppmv; 4He/20Ne = 320) having a moderate 3He/4He ratio (0.44Ra). C1 was enriched relative to other hydrocarbons (C1/(C2+C3) > 3000). The δ13C values for C1 (-40.1‰VPDB), C2 (-28.3‰VPDB), and C5 (-28.0‰VPDB) were similar to those of hydrocarbons produced by thermal decomposition of organic matter. The contribution of the mantle-derived 3He-enriched component in coexisting helium also supports thermogenic generation. On the other hand, the other light hydrocarbons showed an unusual 13C-enrichment in C3 (-19.1‰VPDB), iso-C4 (-22.4‰VPDB), and n-C4 (-19.9‰VPDB). C3 and C4 had been fractionated both chemically and isotopically through subsequent microbial destruction during the long storage from Miocene in the gas reservoir. In addition, the anaerobic oxidation of CH4 within shallow sediments removed about 20% of CH4, until seepage into ocean water column. Regarding the contribution of gases originating from hydrate dissociation to the bubbles, observed helium amount in the bubbles suggests that methane hydrate is considered to be a minor contributor to the bubbles, at least at present. Direct leakage of gases from deep reservoirs is a more plausible for the source. Regarding the fate of the hydrocarbons in the bubbles in the water column, all rising bubbles at Kuroshima Knoll dissolved within 140 m of the seafloor. After the dissolution, the plume spreads horizontally along with the surface of equal density in the water column, while the concentrations decrease through dilution by eddy diffusion rather than by oxidation