The infertility of many couples seems to rest on an enigmatic dysfunction of the men’s sperm, rendering early diagnosis and evidence-based treatment by medically assisted reproduction impossible. Using a novel laboratory test, we assessed the function of the flagellar Ca2+channel CatSper in sperm of almost 2,300 men undergoing a fertility workup. Thereby, we identified a group of men with mutations inCATSPERgenes affecting the function of the channel. Although standard semen and computer-assisted sperm analysis were unremarkable, the couples required intracytoplasmic sperm injection (ICSI) to conceive a child. We show that their seemingly unexplained infertility and need for ICSI is, in fact, due to the failure of CatSper-deficient human sperm to hyperactivate and penetrate the egg coat. In summary, our study reveals that defective CatSper function represents the most common cause of unexplained male-factor infertility known thus far and that CatSper-related infertility can readily be diagnosed, enabling evidence-based treatment.