As emerging contaminants, microplastics (mPS, <5mm) have been reported to adversely affect the plant growth; however, the mechanisms of mPS-induced growth limitation are rarely known. Here, it was found that the plastic particles were absorbed and accumulated in barley plants, which limited the development of rootlets. The mPS-treated plants had significantly higher concentrations of H2O2 and O2- in roots than the control. The mPS significantly increased the activities of dehydroascorbate reductase, glutathione reductase, ADP-Glucose pyrophosphorylase, fructokinase and phosphofructokinase, while decreased the activities of cell wall peroxidase, vacuolar invertase, sucrose synthase, phosphoglucomutase, glucose-6-phosphate dehydrogenase and phosphoglucoisomerase in roots. The changes in activities of carbohydrate and ROS metabolism enzymes in leaves showed a different trend from that in roots. The mPS plants possessed a higher trans-zeatin concentration while lower concentrations of indole-3-acetic acid, indole-3-butyric acid and dihydrozeatin than the control plants in leaves. However, the phytohormone changes in roots were distinct from those in leaves under mPS. In addition, significant correlations between enzyme activities and phytohormone concentrations were found. It was suggested that the phytohormone regulatory network plays key roles in regulating the activities of key enzymes involved in carbohydrate and ROS metabolisms in response to mPS in barley.