The study was aimed to investigate the effect of corn stalk pre-treatment duration on biogas production when cow dung and corn stalk was co-digested in an anaerobic digestion. Corn stalks were pre-treated in different durations: 2-days, 5-days, and 8-days before being added to cow dung into anaerobic co-digesters. The experiments were set up randomly by using triplicate batch anaerobic apparatus in 21 L containers that run in 60-days. The mixing ratio between a corn stalk and cow dung was 50%: 50% (based on the volatile solid value of each material), but corn stalk was cut into small pieces with around 10 cm length, while the cow dung was air dried. The results of the study indicated that all operation parameters such as temperature, pH, and alkalinity in the anaerobic batch were suitable for biogas production. The results showed that there was a significant improvement in total gas produced in the pre-treated 5-days treatment (206.4±8.4 L) compared to 2-days (153.4±9.6 L), and 8-days ones (174±11.1 L). The biogas yield of the pretreated 2-days, 5-days, and 8-days treatments were 392.7±9.8 L/kg VSfermented, 469.8±10.1 L/kg VSfermented and 497.1±13.3 L/kg VSfermented, respectively, that was not significantly different (5%). In all treatments, low concentration of methane in the beginning phase had been observed but increased and reached the optimum value for energy use after 10 days. The result of the study showed that it is preferable to have 5-days pre-treatment of corn stalk before the corn stalk is loaded to an anaerobic digester in combination with cow dung.