The dysregulation of microRNAs (miRNAs) plays an important role in asthenozoospermia. This study evaluated the sperm microRNA‐423‐5p (miR‐423‐5p) expression in asthenozoospermia and normozoospermia, exploring the role of miR‐423‐5p in asthenozoospermia. Eighty participants were divided into asthenozoospermic (AZS, n = 40) and normozoospermic (Norm, n = 40) groups. Fresh semen samples were collected and the sperm cells were separated. Quantitative Real‐Time polymerase chain reaction was used to measure the sperm miR‐423‐5p level. Receiver operating characteristic curve (ROC) was employed to test the diagnostic performance of miR‐423‐5p in asthenospermia. Dual‐reporter luciferase assay was adopted to confirm the target gene of miR‐423‐5p. The target gene level in asthenozoospermia and normozoospermia was measured, and the biological function of target gene in asthenozoospermia was evaluated. Results showed that the miR‐423‐5p expression level in the AZS group was higher than that in Norm group, which was positively correlated with the severity of asthenozoospermia. ROC analysis of miR‐423‐5p showed an area under curve (AUC) of 0.69 (95% confidence interval = 0.57–0.80, p <0 .01), with 80% sensitivity and 60% specificity. Glutathione S‐transferase mu 1 (GSTM1) is a target gene of miR‐423‐5p, which significantly decreased in the AZS group. Compared with Norm group, glutathione S‐transferase (GST) activity and total antioxidant capacity (TAC) level decreased, while malondialdehyde (MDA) level increased in the AZS group. Furthermore, GST activity and TAC level were negatively correlated with miR‐423‐5p expression, while MDA level was positively correlated with miR‐423‐5p expression. In conclusion, the sperm miR‐423‐5p level significantly was upregulated in asthenozoospermia. High‐level miR‐423‐5p inhibited sperm motility through targeting GSTM1 to promote oxidative stress.