Abstract: Sperm contain a complex population of coding and noncoding RNA, and the utility of sperm RNA in fertility research is currently being explored. The aim of this study was to estimate the content of transcripts of protamines 1 and 2 in human ejaculated spermatozoa in relation to semen quality and sperm DNA fragmentation between fertile and infertile patients. Human ejaculates were obtained from 33 patients and semen analyzes were assessed by WHO criteria (2010). We evaluated the sperm DNA fragmentation measured by TUNEL assay. The ejaculates of patients were purified by density-gradient centrifugation, sperm cells were lysed, and mRNA was extracted, reverse transcribed, and subjected to real-time qPCR using specific primer pairs for protamine-1, protamine-2 RNA. The sperm protamine mRNA ratio in normozoospermic men (n = 19; 2.86 ± 0.16) differed significantly from that of patozoospermic patients (n = 14; 3.43 ± 0.22; p < 0.05). A significant correlation was shown between sperm DNA fragmentation and the PRM2/PRM1 mRNA ratio (r = 0.33; p < 0.05). In the group of patients with an increased sperm DNA fragmentation (n = 14; 2.74 ± 0.18), the PRM2/PRM1 ratio was significantly higher than in the group of patients with normal rates (n = 19; 3.26 ± 0.19; p < 0.05). An abnormal sperm protamine ratio was associated with poor semen quality and DNA fragmentation. Finding sperm-quality markers would help to understand the causes of male infertility and to improve male reproductive health.