Methylation of miRNAs at the 2'-hydroxyl group on the ribose at 3'-end (2'-O-methylation, 2'Ome) is critical for miRNA function in plants and Drosophila. Whether this methylation phenomenon exists for mammalian miRNA remains unknown. Through LC-MS/MS analysis, we discover that majority of miR-21-5p isolated from human non-small cell lung cancer (NSCLC) tissue possesses 3'-terminal 2'Ome. Predominant 3'-terminal 2'Ome of miR-21-5p in cancer tissue is confirmed by qRT-PCR and northern blot after oxidation/β-elimination procedure. Cancerous and the paired non-cancerous lung tissue miRNAs display different pattern of 3'-terminal 2'Ome. We further identify HENMT1 as the methyltransferase responsible for 3'-terminal 2'Ome of mammalian miRNAs. Compared to non-methylated miR-21-5p, methylated miR-21-5p is more resistant to digestion by 3'→5' exoribonuclease polyribonucleotide nucleotidyltransferase 1 (PNPT1) and has higher affinity to Argonaute-2, which may contribute to its higher stability and stronger inhibition on programmed cell death protein 4 (PDCD4) translation, respectively. Our findings reveal HENMT1-mediated 3'-terminal 2'Ome of mammalian miRNAs and highlight its role in enhancing miRNA's stability and function.