Poor sperm quality is the major cause of infertility in humans. Other than sex-linked factors, the genetic basis for male infertility is poorly defined, largely due to practical difficulties in studying the inheritance of this trait in humans. As an alternative, we have conducted forward genetic screens in mice to generate relevant models. We report on the identification and characterization of a chemically-induced mutation, Ste5Jcs1, which causes affected male mice to be sterile or subfertile. Mutant sperm exhibited depressed progressive motility associated with a rigid flagellar midpiece (but not principal piece) segment, which could not be rescued by treatment with agents that stimulate cAMP or calcium signaling pathways. Overall mutant sperm ultrastructure appeared normal, including the axoneme, although the midpiece mitochondrial sheath showed abnormal electron density patterns. Positional cloning of Ste5Jcs1 led to the identification of a mutation in a novel gene called Nsun7, which encodes a protein with a Sun domain that is homologous to tRNA and rRNA cytosine methyltransferases. Therefore, Ste5Jcs1 mutation uncovers a previously unrecognized biological process in sperm that underscores the functional compartmentalization of the midpiece and principal piece of the flagellum.