Sumoylation is a post‐translational modification involved in the regulation of several cell functions. Recent studies suggest its involvement in spermatogenesis, but occurrence and function of SUMO (small ubiquitin‐like modifier) in mature spermatozoa remain unknown. We report the occurrence of several SUMO1‐conjugated proteins, in a range of 20–85 kDa, in ejaculated spermatozoa. By cytofluorimetric analysis, we evaluated the percentage of SUMO1‐positive spermatozoa in 58 subjects undergoing semen analysis in our laboratory and correlated the obtained values with semen parameters. We found that the percentage of SUMO1‐positive spermatozoa was inversely correlated with total (r = −0.35, p < 0.01) and progressive motility (r = −0.29, p < 0.05). Such correlations become stricter when only asthenospermic subjects were included in the analysis (r = −0.58, p = 0.01 for progressive motility, n = 17) and were lost in non‐asthenospermic subjects. By immunofluorescence and immunoconfocal fluorescence, we demonstrated that SUMO1 is mainly located in the nucleus and, occasionally, in the midpiece of spermatozoa. Immunoelectron microscopy as well as a long permeabilization protocol demonstrated a massive localization of SUMO‐1 in the nucleus. By using a fluorescent probe to distinguish dead/live cells, we show that SUMO1 is mainly present in live spermatozoa. In conclusion, sumoylation of human spermatozoa may be involved in the regulation of motility.