In a recent genome-wide association study, male reproductive ability was assessed for 7962 Fleckvieh sires (Pausch et al. 2014 PLoS Genet. 10, e1004044). Forty bulls in the population with exceptionally poor reproductive performance were found to be homozygous for a nonsense mutation in the transmembrane protein 95 (TMEM95)-encoding gene. Ejaculates from these individuals exhibited normal sperm concentration, morphology, viability, and motility. However, out of 35,671 inseminations with these bulls, only 1.7% resulted in pregnancies. In addition, none of the bulls with normal reproductive performance was homozygous for this mutation. The aim of this study was to examine the effect of this nonsense mutation in TMEM95 on bovine sperm function in vitro. In all experiments, data were assessed for normality of distribution and analysed using a one-way ANOVA. The model included the main effects of treatment, replicate and their interactions. In Experiment 1, the fertilizing ability of sperm, assessed as oocyte cleavage rate, from 5 homozygous (mt/mt), 3 heterozygous (wt/mt), and 2 wildtype (wt/wt) Fleckvieh bulls was assessed. Oocytes fertilised with sperm from mt/mt males had a lower cleavage rate at 48 h post-fertilization than oocytes fertilised with sperm from wt/wt or wt/mt bulls (26 ± 3%, 72 ± 2%, and 79 ± 3%, respectively; P < 0.01). In addition, the kinetics of cleavage were slower in the mt/mt group as evidenced by a lower proportion of embryos beyond the 2-cell stage at 48 h (32 ± 12.6%, 90 ± 3.6%, and 87 ± 0.4%, respectively, P < 0.01). This translated into a lower blastocyst rate at Day 8 in the mt/mt group in comparison with wt/mt and wt/wt groups (2 ± 1%, 27 ± 3%, and 40 ± 2%, respectively, P < 0.01). In Experiment 2, fluorescent staining of oocytes 3 h after fertilization revealed a lower number of sperm from mt/mt bulls bound to the zona pellucida (ZP) in comparison with sperm from wt/wt or wt/mt individuals (3 ± 1.0, 11 ± 2.3, and 24 ± 9.4, respectively; P < 0.05). In order to further study the potential role of TMEM95 in sperm-ZP interaction, in Experiment 3 sperm from the 3 groups were used to fertilize ZP-free oocytes. Consistent with the previous results, sperm from mt/mt bulls were less able to penetrate oocytes with no ZP than sperm from wt/wt or wt/mt bulls (6 ± 2%, 44 ± 8%, and 68 ± 14%, respectively; P < 0.05). In conclusion, these results suggest that TMEM95 is involved in events that take place before ZP binding but are required for sperm to interact normally with the oocyte vestments and, ultimately, to achieve fertilization. Further study of the possible role of this protein in the initiation of sperm capacitation and hypermotility, as well as in early embryo development, will shed more light regarding the crucial role of TMEM95 in sperm function.