Abstract: The aim of the present study was to investigate the effect of fractions from simulated gastrointestinal digest of aqueous extract of velvet antler (AEVA-SGD) on murine splenocytes proliferation. AEVA-SGD was fractionated by gel chromatography and RP-HPLC consecutively. The effect of each fraction on the proliferation of murine splenocytes was detected by WST-8. The effective fractions were preliminary identified by high resolution mass spectrometry. The results showed that AEVA-SGD was separated into six fractions (A^F) by Sephadex LH-20. Fraction F (Fr. F) stimulated the proliferation of resting murine splenocytes, whereas inhibited the proliferation of ConA-stimulated murine splenocytes. The effect of Fr. F on the proliferation of murine splenocytes was more effective than the other fractions. After separation by RP-HPLC, Fr. F was fractionated into three parts. The main components of each part were preliminary identified as:guanine (Fr.F1) , 1-methylguanine and xanthine (Fr. F2) , a mixture of small peptides (Fr. F3) . Compared with Fr. F1 and Fr. F2, the effect of Fr. F3 on the proliferation of murine splenocyte was more effective. The total effect of Fr. F on the proliferation of murine splenocyte was due to the synergistic effects of its fractions.