The Piwi−interacting RNA (piRNA) pathway is a RNA silencing pathway that represses the expression of genes and transposable elements (TE) in the gonads via binding of piRNAs to their complimentary RNA targets. Mammalian piRNAs are 26−31nt in length and are 2’−O−methylated at their 3’ termini. Although the biogenesis of piRNAs remains unclear, Hen1, a plant microRNA (miRNA) 2’−O−methyltransferase, is known to play an important role in piRNA stabilization via methylating the 2’OH at the 3’ termini of sRNAs in plants. Deletion of hen1 in Zebrafish reduced piRNA content and led to an exonuclease−mediated shortening of the piRNAs that resulted in female sterility. In order to understand the function of mouse HEN1 on male fertility, we have generated a mouse model containing a point mutation in the Hen1 ortholog that produces truncated and unstable Hen1 transcripts. Hen1 homozygous mutant males are sterile. They produce greatly reduced numbers of sperm with round heads, and the epididymides are virtually devoid of sperm. Western and northern blots, demonstrated the presence of more than two HEN1 isoforms, however deletion of a 43kDA isoform leads to the sterility phenotype. Real−time PCR and western blot showed that Hen1 is expressed in the new born testis with its expression peak in day 30 and adult testes, suggesting the highest concentration of Hen1 in spermatids. The loss of HEN1 function results in piRNA instability including pachytene piRNA1. Furthermore, increased Line1 expression was found in Hen1 mutant using real-time PCR, Northern blot and in situ hybridisation. Based on these data, we propose important roles of HEN1 in regulating pachytene piRNA stability, post-natal Line1 repression and the regulation of numerous haploid germ cell mRNAs.