The reproductive success drastically depends upon gametes’ motility. In the case of mammals, the character and speed of spermatozoa movements are determined by flagellum structure and activity. Modifications of structural flagellar proteins, for example, microtubule α-tubulin acetylation, strongly affect stabilization, softening, and flexibility of flagella. The possible impact of other posttranscriptional modifications is less clear. Here we demonstrate the presence of serotonylated proteins (covalent binding of serotonin catalyzed by enzyme transglutaminase) in rodents’ testis and spermatozoa. We combined a modified click-reaction method with propargylated serotonin (5-PT) and azide-biotin or azide-Alexa488 with immunostaining and 3D confocal imaging and performed detailed visualization during rat and mice spermatogenesis. Serotonylated proteins are located in a zone of late spermatids within the testis and in the acrosome and the proximal part of the flagellum of mature isolated spermatozoa. The positive reaction occurs in the same regions after 5-PT click-reaction, immunostaining with anti-5-HT, and anti-N-e(γ-glutamyl) lysine antibodies. Cystamine (transglutaminase inhibitor) decreases the staining signal intensities confirming the transglutaminase-dependent specificity of all labeling. Several flagellar structural proteins demonstrate a high degree of colocalization with 5-PT and anti-5-HT labeling suggesting them as candidate targets for serotonylation. Our finding of serotonylated proteins in specific zones of the mammalian testis and within mature spermatozoa suggested their role in spermatozoa capacitation and mature spermatozoa motility. The work was supported by Russian Science Foundation # 17-14-01353.