This study looked at how methionine (Met) affects on serum biochemical indicators, antioxidant indicators, and the fecal microbiota of lactating donkeys. A total of 18 healthy donkeys at the mid-lactation stage were used in this study. They were given concentrate meals with varying concentrations of Met after being randomly split into three groups: Met group I (M1) received 5 g/d of Met, Met group II (M2) received 15 g/d of Met, and the control group (C) received 0 g/d of Met. The trial lasted for five weeks in total. Fecal microbes, antioxidant markers, and blood biochemical indicators were examined. The findings showed that while the levels of malondialdehyde (MDA) decreased (