In this study, the influence of temperature on biogas production efficiency and the microbial community structure was investigated in a two-phase anaerobic digestion reactor for co-digestion of cow manure and corn straw. The results illustrated that the contents of solluted chemical oxygen demand (SCOD) and volatile fatty acid (VFA) in the acidogenic phase and biogas production in the methanogenic phase maintained relatively higher levels at temperatures ranging from 35–25 °C. The methane content of biogas production could be maintained higher than 50% at temperatures above 25 °C. The microbial community structure analysis indicated that the dominant functional bacteria were Acinetobacter, Acetitomaculum, and Bacillus in the acidogenic phase and Cenarchaeum in the methanogenic phase at 35–25 °C. However, the performances of the acidogenic phase and the methanogenic phase could be significantly decreased at a lower temperature of 20 °C, and microbial activity was inhibited obviously. Accordingly, a low temperature was adverse for the performance of the acidogenic and methanogenic phases, while moderate temperatures above 25 °C were more conducive to high biogas production efficiency.