During the rutting season, stag semen is accompanied by a sticky, dense secretion called yellow fraction (YF). There is little information about the role, biology, physiology, and most importantly, the composition of this fluid. The aim of this study was to isolate and identify zinc ions (ZnBPs) and heparin binding proteins (HBPs) from YF of the red deer ( Cervus elaphus L.). Using liquid chromatography, the presence of 6 fractions of ZnBPs (71, 65, 55, 16, 14 and 12 kDa) and 22 fractions of HBPs (163, 140, 96, 78, 71, 65, 55, 49, 33, 31, 26, 25, 24, 22, 18, 16, 13, 12, 11, 10, 9 and 8 kDa) in YF proteome was demonstrated. By means of two-dimensional electrophoreses and MALDI-TOF/TOF mass spectrometry some of them were then identified. Amongst ZnBPs the following were identified: glutaminyl-peptide cyclotransferase, inhibitor of carbonic anhydrase-like, potassium voltage-gated channel subfamily E member 2, WD repeat-containing protein 38 isoform X4. Amongst the HBPs metalloproteinase inhibitor 2 (TIMP2), seminal plasma glycoprotein PSP-I and adseverin (scinderin) were identified. Identifying all ZnBPs and HBPs present in YF may broaden up-to-date knowledge concerning the biology, physiology and preservation of red deer semen.