Mesenchymal stem cells (MSCs) are used as therapeutic tool for the treatment of immune diseases. The inflammatory environment also influences the characteristics of MSCs after transplantation. The aim of the study was to investigate the effects of pro-inflammatory cytokines on the characteristics of umbilical cord mesenchymal stem cells (UCMSCs). UCMSCs were exposed to pro-inflammatory cytokines in vitro for 3 and 7 days, and the biological properties were analyzed. The results showed that the proliferation ability was suppressed by interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β). The adipogenic capacity was inhibited in all conditioned medium, while the chondrogenic and osteogenic capacity was enhanced by TNF-α and IL-1β in vitro. Prostaglandin E2 (PGE2) was increased by IL-1β on the third day, and angiopoietin-1 (Ang-1) was inhibited appreciably by TNF-α on the seventh day. Interleukin-6 (IL-6) was increased by TNF-α and IL-1β, and hepatocyte growth factor (HGF) was inhibited by all inflammatory cytokines. IFN-γ secretion level from human peripheral mononuclear cells (hPBMCs) was lowered by UCMSCs which had been stimulated by TNF-α or IL-1β for 3 days. Moreover, IFN-γ and TNF-α secretion level was only inhibited by UCMSCs which had been by stimulated IFN-γ for 3 days but not 7 days. Our data demonstrated that different inflammatory cytokines and the duration of treatment had different effects on the properties of UCMSCs, which might be instructive for clinical pretreatment in cellular therapeutics.