The earliest unambiguous evidence for animals is represented by various trace fossils in the latest Ediacaran Period (550-541Ma), suggesting that the earliest animals lived on or even penetrated into the seafloor.Yet, the O-2 fugacity at the sediment-water interface (SWI) for the earliest animal proliferation is poorly defined. The preferential colonization of seafloor as a first step in animal evolution is also unusual. In order to understand the environmental background, we employed a new proxy, carbonate associated ferrous iron (Fe-carb), to quantify the seafloor oxygenation. Fe-carb of the latest Ediacaran Shibantan limestone in South China, which yields abundant animal traces, ranges from 2.27 to 85.43 ppm, corresponding to the seafloor O-2 fugacity of 162 mu mol/L to 297 mu mol/L. These values are significantly higher than the oxygen saturation in seawater at the contemporary atmospheric pO(2) levels. The highly oxygenated seafloor might be attributed to O-2 production of the microbial mats. Despite the moderate atmospheric pO(2) level, microbial mats possibly provided highly oxygenated niches for the evolution of benthic metazoans. Our model suggests that the O-2 barrier could be locally overcome in the mat ground, questioning the long-held belief that atmospheric oxygenation was the key control of animal evolution.