Anaerobic digestion is an effective method for treating animal by-products, generating at the same time green energy as methane (CH4). However, the methods and mechanisms involved in anaerobic digestion of α-keratin wastes like hair, nails, horns and hooves are still not clear. In this study we investigated the feasibility of anaerobically co-digesting ground beef hooves in the presence of swine manure or slaughterhouse sludge at 25 °C using eight 42-L Plexiglas lab-scale digesters. Our results showed addition of beef hooves statistically significantly increased the rate of CH4 production with swine manure, but only increased it slightly with slaughterhouse sludge. After 90-day digestion, 73% of beef hoof material added to the swine manure-inoculated digesters had been converted into CH4, which was significantly higher than the 45% level achieved in the slaughterhouse sludge inoculated digesters. BODIPY-Fluorescent casein staining detected proteolytic bacteria in all digesters with and without added beef hooves, and their relative abundances corresponded to the rate of methanogenesis of the digesters with the different inocula. Fluorescence in situ hybridization in combination with BODIPY-Fluorescent casein staining identified most proteolytic bacteria as members of genus Alkaliphilus in the subfamily Clostridiaceae 2 of family Clostridiaceae. They thus appear to be the bacteria mainly responsible for digestion of beef hooves.