Grapsid crabs are one of the most abundant and potentially the most important group of macrofauna inhabiting mangrove forests. A field study was conducted in the Manko wetland (Okinawa Island, southern Japan) to investigate how the burrowing crab Helice formosensis affects the sedimentary fatty acid (FA) and physicochemical characteristics of subtropical mangrove sediments. Multi-dimensional scaling (MDS) results from FA profiles revealed clear differences between burrow compartments and sediments with and without crabs. The impacts of burrowing were demonstrated by higher percentages of bacterial, vascular plant, and macroalgal FA markers in the burrow compartments and crab sediment areas. Conductivity and redox potential were significantly higher in sediments of the burrow opening shaft than in the burrow chamber. We found a similar pattern in surface sediments with crabs, but not in surface sediments without crab habitats. These results suggest that H. formosensis significantly influences the physicochemical properties, FA composition, and organic matter profiles of its surrounding environment.