Ca2+ plays a central role in the regulation of sperm motility. We recently reported an unexpected role of CNNM4, a Mg2+ transporter, in this process by demonstrating perturbed Ca2+ influx and gradual loss of motility of Cnnm4-deficient sperm. However, Cnnm4-deficient male mice were not entirely infertile, and a significant Ca2+ response was still observed in their sperm. In the present study, we generated Cnnm4-deficient mice harboring a non-functional Cnnm2 allele (Cnnm2(Delta)), to examine whether CNNM2 compensates for the lost function of CNNM4 in sperm. Cnnm2+/(Delta); Cnnm4(Delta/Delta) mice were infertile, and no obvious histological abnormalities were noted in their testis and epididymis. Their sperm showed normal morphology, but became immotile much more rapidly than those from Cnnm4(Delta/Delta) mice. When capacitation was initiated using serum albumin application, a rapid increase of intracellular Ca2+ levels was observed in most wild-type sperm, but only about half of sperm from Cnnm4(Delta/Delta) mice exhibited a Ca2+ response, and the response rate was further reduced in sperm from Cnnm2(+/Delta); Cnnm4(Delta/Delta) mice. Thus, sperm motility and Ca2+ response were more severely affected in sperm from Cnnm2(+/Delta); Cnnm4(Delta/Delta) mice than in those from Cnnm4(Delta/Delta) mice, implicating CNNM2 in regulating these processes. (C) 2016 Elsevier Inc. All rights reserved.