The main objective of this study was to investigate the impact of adding oregano essential oil (OEO) to adapted and unadapted rumen cultures on methane gas (CH4) production and rumen fermentation under in vitro condition. Rumen fluid for culture fermenters was collected from a Holstein cow located in Southern Illinois University farms.The adapted and unadapted rumen cultures were obtained from continuous culture fermenters fed a control diet, or control diet plus OEO at 250 mg day-1 for 10 days. After 10 days of adaptation, the fermenter’s contents were incubated in a 24 h gas production experiment. Treatments were unadapted culture, unadapted culture plus OEO, adapted culture and adapted culture plus OEO. The OEO was added to rumen cultures at the rate of 500 mg L-1. After 24-h of incubation, total gas production decreased (p<0.03) with the addition of OEO in both cultures and total gas production tended to be lower when added to the adapted cultures. As a percentage of total gas, CH4 production decreased (p<0.05) with OEO addition only in the adapted cultures. Total VFA and acetate concentrations were lower (p<0.05) in the adapted than unadapted cultures and their concentrations decreased (p<0.05) with the addition of OEO, particularly when added to the adapted cultures. Propionate concentrations were also lower (p<0.05) in the adapted than the unadapted cultures and concentrations decreased (p<0.05) with the addition of OEO. The greater effect of OEO on CH4 production in the adapted cultures might have resulted from lower microbial fermentation activity which also might have caused a shift in microbial fermentation favoring less CH4 formation in these cultures.