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Asthenozoospermia (AZS) is characterized by reduced sperm motility, and its pathogenesis remains poorly understood. Piwi-interacting RNAs (piRNAs) have been recognized to play important roles in spermatogenesis. However, little is known about the correlation of piRNAs with AZS. In this study, small RNA sequencing was performed on samples from AZS patients and fertile controls. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to validate the small RNA-seq results. Bioinformatics analyses were performed to predict the functions of differentially expressed piRNAs. Logistic regression models were constructed, and receiver operating characteristic curve (ROC) analysis was used to evaluate their diagnostic performance. A total of 114 upregulated and 169 downregulated piRNAs were detected in AZS patients. GO and KEGG analyses showed that the differentially expressed piRNAs were mainly associated with transcription, signal transduction, cell differentiation, metal ion binding and focal adhesion. These results were verified by qRT-PCR of 8 selected piRNAs. Among the differentially expressed piRNAs tested, piR-hsa-32694, piR-hsa-26591, piR-hsa-18725, piR-hsa-18586 and piR-hsa-2912 produced qRT-PCR results consistent with the sequencing results in AZS compared with controls in the first cohort, whereas the other three genes did not show significant differences in expression. piR-hsa-32694, piR-hsa-26591, piR-hsa-18725, and piR-hsa-18586 were significantly upregulated in AZS. The diagnostic power of the four piRNAs was further analysed using ROC analysis; piR-hsa-26591 exhibited an area under the ROC curve (AUC) of 0.913 (95% CI: 0.795-0.994). Logistic regression modelling and subsequent ROC analysis indicated that the combination of the 4 piRNAs reached good diagnostic efficacy (AUC: 0.935).