Alkaline (especially pH 10) anaerobic fermentation of waste activated sludge (WAS) has been reported to be an effective approach for hydrogen production through inhibiting the homoacetogenesis and methanogenesis. However, the potential effect of the widespread microplastics in sludge on the performance of hydrogen production has never been reported. To fill this knowledge gap, the dominant polyethylene terephthalate (PET) microplastics in WAS were selected as the model microplastics to evaluate their influences on hydrogen production during alkaline anaerobic fermentation of WAS as well as the key mechanisms involved. Experimental results demonstrated that hydrogen production from WAS decreased in the presence of PET microplastics (i.e., 10, 30 and 60 particles/g-TS) compared to the control, with the hydrogen yield at 60 particles/g-TS being only 70.7 ± 0.9% of the control. Although the hydrogen consumption (i.e., homoacetogenesis and methanogenesis) was restrained under alkaline (pH 10) condition, PET microplastics inhibited hydrolysis, acidogenesis and acetogenesis in alkaline WAS anaerobic fermentation, leading to the inhibitory effect on hydrogen production. This was further confirmed by the microbial analysis, which clearly showed PET microplastics caused the shift of the microbial community toward the direction against hydrolysis-acidification. Mechanism studies revealed that PET microplastics carried on their negative influence mainly through leaching the toxic di-n-butyl phthalate (DBP). The reactive oxygen species (ROS) and live/dead staining tests indicated that the increased ROS was induced by PET microplastics, causing more cells dead, which further resulted in the decreased production of hydrogen.