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Perfluorooctanoic acid (PFOA) that accumulates in wastewater and excess sludge interact with the anaerobes and deteriorate the energy recovery and pollutants removal performance in the anaerobic digestion (AD) system. However, the interaction between PFOA and microbial metabolism in the AD systems remains unclear. This study aimed to clarify the effects and mechanism of PFOA on the AD process as well as the removal pathways of PFOA in an AD system. The results showed that the methane recovery efficiency was inhibited by 7.6–19.7% with the increased PFOA concentration of 0.5–3.0 mg/L, and the specific methanogenesis activity (SMA) was inhibited by 8.6–22.3%. The electron transfer system (ETS) was inhibited by 22.1–37.3% in the PFOA-containing groups. However, extracellular polymeric substance (EPS) gradually increased due to the toxicity of PFOA, and the ratio of protein to polysaccharide shows an upward trend, which led to the formation of sludge aggregates and resistance to the toxic of PFOA. The PFOA mass balance analysis indicated that 64.2–71.6% of PFOA was removed in the AD system, and sludge adsorption was the main removal pathway, accounting for 36.1–61.2% of the removed PFOA. In addition, the anaerobes are proposed to have the potential to reduce PFOA through biochemical degradation since 10.4–28.2% of PFOA was missing in the AD system. This study provides a significant reference for the treatment of high-strength PFOA-containing wastes. [ABSTRACT FROM AUTHOR]