We investigate the site amplification characteristics in the 0.2–10 Hz frequency band at the DONET1 seafloor observation network, which is installed in the Nankai subduction zone in Japan, using a spectral inversion methodology from observed ground‐motion data. The spectral inversion indicates that the seafloor stations have strong site amplification comparable with or larger than that at land stations in a large basin. The site amplification characteristics significantly differ among the stations, which is likely to reflect the spatial variation in the underground structure beneath DONET1, such as an accretionary wedge and shallow soft sediments. Near‐land stations, which are located above the Kumano trough and the thick accretionary wedge, have a much larger site amplification factor in the broadband frequency range, and their peak values reach . Stations located near the trench axis have a remarkably large site amplification factor at a frequency greater than 2 Hz, with a sharp peak over 30, whereas the low‐frequency () levels are less than those at other stations. The site amplification characteristics investigated in this study are expected to be useful in evaluating ground motions at seafloor stations and in improving the real‐time forecast of ground motions for subduction earthquakes.