Serpentinite-hosted hydrothermal systems have attracted considerable attention as sites of abiotic organic synthesis and as habitats for the earliest microbial communities. Here, we report a systematic isotopic study of a new serpentinite-hosted system: the Hakuba Happo hot spring in the Shiroumadake area, Japan (36° 42′ N, 137° 48′ E). We collected water directly from the hot spring from two drilling wells more than 500 m deep; all water samples were strongly alkaline (pH> 10) and rich in H 2 (201–664 μmol/L) and CH 4 (124–201 μmol/L). Despite the relatively low temperatures (50–60° C), thermodynamic calculations suggest that the H 2 was likely derived from serpentinization reactions. Hydrogen isotope compositions for Happo# 1 (Happo# 3) were found to be as follows: δ DH 2=− 700‰(− 710‰), δ D-CH 4=− 210‰(− 300‰), and δ DH 2 O=− 85‰(− 84‰). The carbon isotope compositions of methane from Happo# 1 and# 3 were found to be δ C 13=− 34.5‰ and− 33.9‰, respectively. The CH 4–H 2–H 2 O hydrogen isotope systematics indicate that at least two different mechanisms were responsible for methane formation. Happo# 1 has a similar hydrogen isotope compositions to other serpentinite-hosted systems reported previously. The elevated δ D-CH 4 (with respect to the equilibrium relationship) suggests that the hydrogen of the Happo# 1 methane was not sourced from molecular hydrogen but was derived directly from water. This implies that the methane may not have been produced via the Fischer–Tropsch-type (FTT) synthesis but possibly by the hydration of olivine. Conversely, the depleted δ D-CH 4 (with respect to the equilibrium relationship) in Happo# 3 suggests the incorporation of biological methane. Based on a comparison of the hydrogen isotope systematics of our results with those of other serpentinite-hosted hydrothermal systems, we suggest that abiotic CH 4 production directly from H 2 O (without mediation by H 2) may be more common in serpentinite-hosted systems. Hydration of olivine may play a more significant role in abiotic methane production than previously thought.