For decades, it was widely accepted that mammalian spermatozoa were devoid of translational activity due to the functional and morphological changes that occurred during spermatogenesis. However, it was proved that capacitated spermatozoa possess translation activity, which appears to be ensured by mitochondrial ribosomes. Since mitochondrial translation has several particularities, the hypothesis that sperm translation occurs through mitochondrial ribosomes raises some questions. The main objectives of this work were to study the impact of translation inhibition in the bovine spermatozoa proteome and to identify the de novo synthesized proteins by capacitated bovine spermatozoa. To identify which proteins were de novo translated, capacitated bovine spermatozoa were treated with puromycin and puromycin-peptides were immunoprecipitated. To study the impact of translation inhibitors in capacitated sperm proteome, bovine spermatozoa were capacitated in the presence or absence of translation inhibitors - Cycloheximide (CHX, cytoplasmic translation inhibitor) and D-Chloramphenicol (D-CP, mitochondrial translation inhibitor). After capacitation, sperm lysates were analysed by mass spectrometry. We identify nine newly synthesised cytoplasmic proteins by capacitated bovine spermatozoa, which are related to sperm-specific processes. Both translation inhibitors impact differently the sperm proteome leading to the deregulation of cytoplasmic protein related to sperm physiology and function. These results support the existence of cytoplasmic translation in bovine spermatozoa during capacitation.