The Mugi mélange in the Shimanto accretionary complex, southwest Japan, records faulting and fluid flow patterns at the updip limit of the seismogenic region of the Nankai subduction zone. To characterize the origin and behavior of syn-tectonic fluids, we investigated the carbon, oxygen, and strontium isotopic compositions, and rare earth element (REE) patterns of syn-tectonic calcite within veins along fault zones in the mélange, as well as the Sr isotopic compositions and REE patterns of surrounding host rocks. With the exception of intra-basalt veins formed prior to subduction, the δ13C values of veins range from −10‰ to −19‰, suggesting a mixed carbon source (i.e., marine carbonate and organic matter). The vein-forming fluids have positive oxygen isotopic compositions (+2‰ to +9‰ (SMOW)) and high 87Sr/86Sr values (0.70794–0.70850), suggesting that the source was rock-buffered fluids affected both by terrigenous sediments and altered oceanic crust. The veins found in filling the fault zone associated with tectonic underplating have different REE patterns to those of the other veins, implying a difference in physicochemical processes affecting the fault zone near the subduction megathrust.