Wastewater containing N, N-dimethylformamide (DMF) was treated by artificially mixing the anaerobic granular sludge (AGS) with DMF-degrading activated sludge (DAS) in this study. An up-flow anaerobic sludge blanket (UASB) successfully treated wastewater containing approximately 2000 mg L-1 DMF during an operation period of 215 days. An inoculation of DAS brought about remarkable results: a rapid start-up with effective DMF methanogenic degradation on the first day, and under a low organic loading rate (OLR) of 1.63-4.22 g COD L-1 day-1, the UASB maintained excellent DMF removal efficiency at over 96% along with the high methane production rate (MPR). However, when the OLR increased to 9.24 g COD L-1 day-1, DMF removal efficiency and MPR dropped to 47.36% and 1.05 L L-1 day-1. A further increase in the OLR to 13.25 g COD L-1 day-1 resulted in a sharp deterioration in the DMF-degrading ability, at merely 19.19% and a low MPR of 0.38 L L-1 day-1. The excessive elevation of OLR resulted in the insufficient hydrolysis of the DMF, and the further weakening of the conversion from DMF to intermediates and an acceleration the decaying of DMF-hydrolyzing bacteria. Methane-producing archaea was starved of intermediates when hydrolysis was inadequate. Since the DAS can be massively domesticated, and the OLR should be kept lower than 6.17 g COD L-1 day-1, the timely replenishing of the DAS to the UASB may be a solution to maintain a stable and effective DMF hydrolysis for long-term operation. The results of this study provide insight for the development of a new concept and an improved method for the effective treatment of wastewater containing degradation-resistant organics.