The effects of substrate mingling ratio (SMR) (1:1, 1:2, 1:3, 3:1, and 2:1) and organic loading rate (OLR) (50–90 g total solids per liter per day) on anaerobic co-digestion performance and microbial characteristics were investigated for pig manure (PM) and pretreated/untreated corn stover in batch and semi-continuous anaerobic digestion (AD) system. The results showed that SMR and pretreatment affected co-digestion performance. The maximum cumulative methane yield of 428.5 ml⋅g−1 (based on volatile solids (VS)) was obtained for PCP13, which was 35.7% and 40.0% higher than that of CSU and PM. In the first 5 days, the maximum methane yield improvement rate was 378.1% for PCP13. The daily methane yield per gram VS of PCP13 was 11.4%–18.5% higher than that of PCU13. Clostridium_sensu_stricto_1, DMER64, and Bacteroides and Methanosaeta, Methanobacterium, and Methanospirillum had higher relative abundance at the genus level. Therefore, SMR and OLR are important factor affecting the AD process, and OLR can affect methane production through volatile fatty acids.