Methane production is the main gas energy loss in ruminants and generates a powerful greenhouse gas that contributes to climate changes. Macleaya cordata is a plant commonly utilized additive in livestock diet as it contains various chemical compounds with beneficial health effects. This study aimed to explore the influence of M. cordata extract on in vitro methanogenesis and rumen fermentation. Three cannulated Jingjiang cattle were used as rumen fluid donors. The mixture of rumen fluid and a buffer (60 ml, 1:2 volume ratio) was treated with 6 different concentrations of M. cordata extract (0.01%, 0.11%, 0.21%, 0.31%, 0.41%, and 0.51%) and incubated for 12 and 24 hr. The control sample, with no addition of plant extract, was also analyzed. At each time point, pH, total gas, methane, dry matter (DM) digestibility, neutral detergent fiber (NDF), acid detergent fiber (ADF), ammonia nitrogen (NH3‐N), microbial protein (MCP), and volatile fatty acids (VFA) concentrations were determined. Total gas production decreased with increasing the amount of M. cordata extract at all time points. Methane production also decreased dose‐dependently with M. cordata extract after 3, 6, 9, and 12 hr of incubation, but increased after 24 hr. M. cordata extract decreased the concentration of NH3‐N and VFA, and the amount of acetic, propionic, and butyric acid. M. cordata extract decreased the MCP concentration after 12 hr, but its level returned to the initial value after 24 hr. Supplementation with 0.01, 0.11, and 0.21% of M. cordata extract did not affect the DM digestibility. However, supplementation with 0.31, 0.41, and 0.51% of M. cordata extract significantly decreased the DM digestibility. Supplementation with 0.11% of M. cordata extract effectively reduced methane production without affecting the DM digestion in vitro. However, its effect on in vivo methane production, rumen fermentation, and ruminant production requires further investigation.