Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), is a long non-coding RNA localized in the cell nucleus, known for its multifunctional roles, including potential involvement in spermatogenesis. This study investigates the mechanism by which MALAT1 dysregulation contributes to the pathogenesis of idiopathic non-obstructive azoospermia (iNOA). We analyzed MALAT1 levels in two gene expression profiling datasets comprising patients with obstructive azoospermia (OA) who have normal spermatogenesis and 13 patients with iNOA. The dysregulation of MALAT1 along with the expression levels of its negatively correlated genes were confirmed in a larger cohort of 24 OA patients and 38 iNOA patients. We examined the effects of MALAT1 overexpression in primary human spermatogonial stem cells (SSCs) and Sertoli cells. Additionally, we assessed DNA methylation, as well as levels of H3K27me3 and H3K27Ac level near the etv5 promoter region using ChIP-qPCR. We observed that MALAT1 was overexpressed in testes of iNOA patients with its levels negatively correlating with six spermatogenesis related genes and positively correlated with three others. Overexpression of MALAT1 in SSCs repressed proliferation and induced apoptosis while also suppressing ETS variant transcription factor 5 (ETV5) expression by promoting H3K27 tri-methylation of the ETV5 promoter. Overexpression of MALAT1 in Sertoli cells did not induce apoptosis but impaired their cell supporting function. In conclusion, MALAT1 overexpression in SSCs contributes to the pathogenesis of iNOA via downregulating ETV5 expression and promoting cell apoptosis.