The voicing contrast for Japanese word-initial stops is primarily realized as differences in the Voice Onset Time (VOT). However, previous studies have pointed out that VOT alone cannot differentiate the two stop categories because their VOT values overlap. A few studies have attempted to find another characteristic by examining post-stop fo and voice quality but have failed to find an answer. This study investigated VOT and post-stop fo using data drawn from eighty-two native speakers from four regions. The result shows that post-stop fo differences can distinguish the two categories, but not alone. The acoustic space of VOT and post-stop fo demonstrates a clear distinction between the two categories, while the effect of VOT and post-stop fo on voicing contrast differs from region to region. The result of this study provides evidence that post-stop fo functions as an acoustic characteristic responsible for voicing contrast along with VOT.