The aim of this study was to evaluate the influence of feeding with food and water containing chlorocholinechloride (CCC) on the fertility of male mice in a two-generation study. For this purpose the number of testicular spermatozoa and the relative proportion of primary and secondary spermatocytes involved in spermatogenesis were measured. Furthermore, the fertility of epididymal spermatozoa from tested male mice was investigated in a special in-vitro fertilization system. The experimental food was composed of CCC-treated wheat in the first experiment and CCC-free wheat and water mixed with pure CCC in the second experiment. The CCC residue content in the treated food and water was 0.21 mg/kg and 0.2 mg/L, respectively. Under the influence of feeding with CCC-treated wheat (Experiment 1) the fertilization and cleavage rates of oocytes incubated with spermatozoa from CCC-fed mice were reduced: the fertilization rate 65.1% vs. 21.1% and the cleavage rate 51.9% vs. 20.3%, p < 0.01 (control feeding vs. CCC feeding, respectively). Feeding of sperm donors with pure CCC mixed with untreated wheat pellets or water (Experiment 2) led to a reduction in the fertilization and cleavage rate (control: 60.8%, 32.4%; CCC-food: 29.8%, 12.1%; CCC-water: 30.1%, 10.2%; CCC-food/water: 36.6%, 12.5%; p < 0.01, respectively). The normal course of spermatogenesis was unchanged after the exposure to CCC. Testicular weight, the number of spermatozoa, and the proportion of haploid, diploid, and tetraploid testicular cells were not influenced. However, the functional competence of epididymal spermatozoa from CCC-fed donors was reduced, resulting in a significantly diminished fertilization and cleavage rate in vitro. The results suggest that CCC could interfere with epididymal protein secretion and the process of sperm maturation during passage through the epididymis.