<jats:title>Abstract</jats:title><jats:sec><jats:title>Background</jats:title><jats:p>Lactate dehydrogenase C (LDHC) is specifically expressed in male germ cells and plays critical roles in glycolysis. Glycolysis is required to supply energy for sperm motility. Previous studies showed that <jats:italic>Ldhc</jats:italic> knock‐out mice exhibit impaired sperm motility.</jats:p></jats:sec><jats:sec><jats:title>Objectives</jats:title><jats:p>We established human <jats:italic>LDHC</jats:italic> knock‐in (<jats:italic>hLDHC</jats:italic> KI) mice and examined whether <jats:italic>hLDHC</jats:italic> KI mice can be used to assess LDHC‐targeting drugs.</jats:p></jats:sec><jats:sec><jats:title>Material and methods</jats:title><jats:p><jats:italic>HLDHC</jats:italic> was knocked‐in to the mouse <jats:italic>Ldhc</jats:italic> (<jats:italic>mLdhc</jats:italic>) allele using the CRISPR/Cas9 system. Mating tests, sperm motility examinations with a computer‐assisted sperm analysis (CASA) system, and in vitro fertilization (IVF) were performed. Furthermore, the effect of an LDH inhibitor was analyzed with CASA and IVF.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>HLDHC was detected at the protein level in <jats:italic>hLDHC</jats:italic> KI spermatozoa. <jats:italic>hLDHC</jats:italic> KI mice exhibited comparable sperm motility and male fertility to wild‐type (WT) mice. When we performed IVF using the LDH inhibitor more specific to hLDHC than mLDHC, fertilization rates were reduced in <jats:italic>hLDHC</jats:italic> KI mice but not in WT mice.</jats:p></jats:sec><jats:sec><jats:title>Discussion and conclusion</jats:title><jats:p>Our results reveal that <jats:italic>hLDHC</jats:italic> can rescue the absence of <jats:italic>mLDHC</jats:italic>. Differences in the effect of the LDH inhibitor between WT and <jats:italic>hLDHC</jats:italic> KI mice indicate that <jats:italic>hLDHC</jats:italic> KI mice can be a good model to assess hLDHC inhibitors for preclinical contraceptive studies.</jats:p></jats:sec>