Floating tests were conducted in anaerobic digestion with different OLR of corn stover to investigate formation of floating layers and to find proper agitation interval for preventing floating layer formation. Floating layers were formed in the early stage of no-agitation period. The daily biogas production was decreased by 81.87-87.90% in digesters with no agitation and feeding compared with digesters having agitation. Reduction of biogas production was mainly attributed to poor contact of substrate-microorganisms. Agitation intervals of 10 h, 6 h, and 2 h were found to be proper for eliminating floating layer at OLR of 1.44, 1.78 and 2.11 g(TS) L-1 d(-1), respectively. The proper agitation interval was further validated by anaerobic experiments. It showed that proper agitation interval could not only prevent floating layer formation and achieve high biogas production but also increase energy efficiency of anaerobic digestion. The finding is useful for operating anaerobic digester with corn stover in a cost-effective way. (C) 2015 Elsevier Ltd. All rights reserved.