Non-obstructive azoospermia (NOA) is a common cause of male infertility, and no specific diagnostic indicators exist. In this study, we used human testis datasets GSE45885, GSE45887, and GSE108886 from GEO database as training datasets, and screened 6 signature genes (all lowly expressed in the NOA group) using Boruta algorithm and Lasso regression: C12orf54, TSSK6, OR2H1, FER1L5, C9orf153, XKR3. The diagnostic efficacy of the above genes was examined by constructing models with LightGBM algorithm: the AUC (Area Under Curve) of both ROC and Precision-Recall curves for internal validation was 1.0 (p < 0.05). For the external validation dataset GSE145467 (human testis), the AUC of its ROC curve was 0.9 and that of its Precision-Recall curve was 0.833 (p < 0.05). Next, we confirmed the cellular localization of the above genes using human testis single-cell RNA sequencing dataset GSE149512, which were all located in spermatid. Besides, the downstream regulatory mechanisms of the above genes in spermatid were inferred by GSEA algorithm: C12orf54 may be involved in the repression of E2F-related and MYC-related pathways, TSSK6 and C9orf153 may be involved in the repression of MYC-related pathways, while FER1L5 may be involved in the repression of spermatogenesis pathway. Finally, we constructed a NOA model in mice using X-ray irradiation, and quantitative Real-time PCR results showed that C12orf54, TSSK6, OR2H1, FER1L5, and C9orf153 were all lowly expressed in NOA group. In summary, we have identified novel signature genes of NOA using machine learning methods and complete experimental validation, which will be helpful for its early diagnosis.