Advanced breast cancer remains clinically challenging due to its resistance to chemotherapy. To understand the underlying mechanisms of resistance and identify drugable target, the involvement of ceramide metabolism is investigated. Ceramide levels in breast cancer tissues derived from 30 patients with stage IV breast cancer before and after chemotherapy were analyzed using liquid chromatography mass spectrometry. mRNA and protein levels of ceramide enzymes were examined using western blot and QRT-PCR. The effects of ceramide analog were investigated using cellular assays and xenograft tumor model. The results demonstrated that pro-apoptotic ceramide was significantly lower in all patients after chemotherapy, suggesting that downregulation of ceramide is a common feature of breast cancer patients in response to chemotherapy. Molecular characteristics analysis of ceramide indicated C16:0 as the predominant sphingolipid regulated by chemotherapy in breast cancer patients. Mechanistically, ceramide levels were suppressed by chemotherapy via increasing mRNA and protein levels of UDP-glucose ceramide glucosyltransferase (UGCG). Importantly, inhibition of UGCG using siRNA or upregulation of cellular ceramide levels using C2 ceramide alone inhibited proliferation and induced apoptosis of breast cancer cells, and enhanced the inhibitory effects of chemotherapeutic drugs in vitro and in vivo. This study clearly demonstrated that the decreased ceramide production via up-regulating UGCG was involved in the resistance of breast cancer cells to chemotherapy. Stimulating ceramide or decreasing UGCG can potentially be useful for breast cancer treatment.