SummaryEvidence has been produced that macrophages can actively generate endocannabinoids (eCBs) in response to inflammatory stimuli. AseCBs are involved in the control of several physiological processes, including reproduction, here, we explored whether seminal levels of theeCBs,N‐arachidonoylethanolamine (AEA), and 2‐arachidonoylglycerol (2‐AG), were higher in the presence of leukocytospermia, and were correlated with semen concentration of macrophages. The content ofAEAand 2‐AGwas measured by high‐performance liquid chromatography/mass spectrometry in seminal plasma of ejaculates from 18 leukocytospermic patients (>1 × 106leukocytes/mL) and 21 normozoospermic controls. In the same ejaculates, round cells were phenotyped by flow‐cytometry as leukocytes (CD45+), macrophages (CD14+), and activated macrophages (CD14+,HLA‐DR+). The levels of 2‐AG, but not ofAEA, were significantly higher in ejaculates from leukocytospermic patients than in controls and exhibited a significant correlation with semen concentration of macrophages and activated macrophages. Significant associations of 2‐AGwith macrophages and activated macrophages persisted after adjustment for semen volume and sperm concentration. In conclusion, here we provide evidence that seminal plasma levels of 2‐AGare higher in the presence of leukocytospermia, as a marker of macrophages activation. Further studies are warranted to elucidate possible clinical implications.