We present a pilot study based on a multi-level molecular profiling of semen from 33 healthy young subjects residing in a high (n=18) and low (n=15) polluted area in Italy. Samples were analyzed for spermiogram parameters, protamine/histone ratio, Sperm Nuclear Basic Proteins (SNBP) and DNA interaction, oxidative DNA damage, trace metals in semen, and spermatocyte small-noncoding RNA expression. Although comparable mobility and concentration of sperm cells were observed between samples from the two areas, a higher immature/immune cell concentration characterized the semen of subjects from the high-polluted area. These subjects also showed a lower ratio of protamine to histone and a reduced ability of SNBP to bind and protect DNA from oxidative damage. Trace metal analysis showed an altered ratio of copper to zinc in their semen. In spermatocytes from the high-polluted area, altered levels of 32 microRNAs involved in intraflagellar transport, response to oxidative species, nuclear envelope organization, and lipid metabolism were observed. These miRNA profiles resemble those of spermatocytes undergoing differentiation, including a decrease of miR-23b-3p and let-7f-5p and an increase of miR-34b-3p levels. MicroRNAs expression profiles were coherently related to the sperm concentration in both study groups. In addition, a significant decrease of piRNA levels was also observed. These results support a model that provides new insights into the environmental impact on male germ cells. Overall design: Thirty-three boys were involved, distributed and residing in two areas, with different environmental pressures based on environmental data from ARPA Campania of which one with high environmental impact in LF municipalities and one with low environmental impact in Upper Middle Sele municipalities in the province of Salerno (namely, Valley of the Sele, VSL). The inclusion criteria used in this project were the following: boys between the ages of 18 and 22 years; healthy; non-drinkers, non-regular (daily) alcohol users (≤ 5 alcoholic units per week); not drug users; not previous oncological diseases and/or chemo/radiotherapy; body mass index (BMI) between 18.5 and 30.5; abdominal circumference <102 cm. After 3-4 days of sexual abstinence, human sperm was collected at the time of morning masturbation and stored in sterile containers. In line with the WHO recommendations (WHO Laboratory Manual for the Examination and Processing of Human Semen, 6th edn. Geneva: World Health Organization, 2021), regular tests were performed on sperm samples to assess their quality (volume, pH, sperm concentration, total and progressive motility, morphology, and round cell concentration).