The aim of this study was to investigate the effect of different levels of aflatoxin on the gas production, in vitro fermentation parameters, dry matter and organic matter digestibility of sheep's diet. This experiment was conducted in a completely randomized design with 5 treatments and 4 replicate per treatment. The treatments included 1.diet without aflatoxin and methanol (control), 2.control with methanol, 3.diet containing 400 ng / ml of aflatoxin B₁, 4.diet containing 800 ng / ml of aflatoxin B₁, and 5.diet containing 1200 ng / ml of aflatoxin B₁ in ruminal fluid. Treatments were incubated at 0, 12, 24, 36, 48, 60, 72, 84, 96 hours and the amount of gas produced was measured using a barometer at 39 °C. The results showed that treatments with high levels of aflatoxin, especially 1200 ng / ml rumen fluid, significantly decreased gas production compared to those without aflatoxin. Addition of different levels of aflatoxin significantly decreased dry matter and organic matter digestibility, microbial mass production, and biodegradability index compared to control group. The results of this study showed that rumen microorganisms have the ability to neutralize aflatoxin at low levels and fermentation conditions are not significantly affected. With increasing level of aflatoxin in the diet, the gas production, digestibility of dry matter and organic matter, and also microbial mass production and PF was reduced. Therefore, it can be concluded that aflatoxin modifies ruminal fermentation processes with reduced nutrient degradability, which is dependent on aflatoxin concentration.