U6 snRNA, as an essential component of the catalytic core of the pre-mRNA processing spliceosome, is heavily modified post-transcriptionally, with 2′-O-methylation being most common. The role of these modifications in pre-mRNA splicing as well as their physiological function in mammals have remained largely unclear. Here we report that the La-related protein LARP7 functions as a critical cofactor for 2′-O-methylation of U6 in mouse male germ cells. Mechanistically, LARP7 promotes U6 loading onto box C/D snoRNP, facilitating U6 2′-O-methylation by box C/D snoRNP. Importantly, ablation of LARP7 in the male germline causes defective U6 2′-O-methylation, massive alterations in pre-mRNA splicing, and spermatogenic failure in mice, which can be rescued by ectopic expression of wild-type LARP7 but not an U6-loading-deficient mutant LARP7. Our data uncover a novel role of LARP7 in regulating U6 2′-O-methylation and demonstrate the functional requirement of such modification for splicing fidelity and spermatogenesis in mice.