Anaerobic digestion of lignocellulosic biomass faces changes such as biomass floating and effluent discharge. To overcome these challenges, a unique removable cartridge anaerobic digester was built and tested using corn stover as the feedstock. Three operating methods differing in the number of cartridges and days of rotation were tested. The first method used three cartridges, with each cartridge being rotated every 7 days. The second and third methods employed four cartridges, with cartridges being rotated every 7 and 9–10 days, respectively. The retention time for methods 1, 2, and 3 was 21, 28, and 38 days, respectively. After observation spanning 1 year, it was found that the cartridge digester was capable of generating a stable amount of biogas for energy without biomass floating or effluent discharging issues. The average daily methane yield from each method was 7.57, 7.11, and 6.82 L/day/kg-VS, and the cumulative methane yield was 158.95, 199.04, and 259.00 L/kg-VS, respectively. Ammonium nitrogen and pH values were in normal ranges throughout the experiment. This study provided new knowledge in operating and optimizing this cartridge digester, which may be broadly used for the anaerobic digestion of lignocellulosic biomass in the near future. Supplementary Information The online version contains supplementary material available at 10.1007/s12155-021-10252-w.