Sulfur isotopic signatures of pyrite (δ34Spy) sensitively react to local environmental change. Here we elucidate mechanism of δ34Spy variation in the Holocene sedimentary strata of the Yangtze River Delta, through the analyses of multiple geochemical and physical indicators including total organic carbon, total nitrogen, stable isotopes of organic carbon, different iron solid phases, Sr/Ba ratios, and changes in sedimentation rates. Two heavy δ34Spy (∼30‰) layers that formed under contrasting redox conditions are preserved in the early Holocene transgressive tidal flat and late Holocene regressive mouth bar facies, respectively. Two hypotheses are proposed to explain these heavy δ34Spy layers: 1) in the transgression process, the heavy δ34Spy layers formed under stronger reducing conditions due to methane leakage; 2) in the regression process, the heavy δ34Spy layers formed under relatively weaker reducing conditions due to rapid deposition and low salinity. This study emphasizes the importance of physicochemical condition characterization in sediments, including redox conditions, in distinguishing the mechanisms that act to produce heavy δ34Spy signals.