5-Fluorouracil (5-FU)is a chemotherapeutic drug widely used to treat colorectal cancer. 5-FU is known to gradually lose its efficacy in treating colorectal cancer following the acquisition of resistance. We investigated the mechanism of 5-FU resistance using comprehensive lipidomic approaches. We performed lipidomic analysis on 5-FU-resistant (DLD-1/5-FU) and -sensitive (DLD-1) colorectal cancer cells using MALDI-MS and LC-MRM-MS. In particular, sphingomyelin (SM) species were significantly up-regulated in 5-FU-resistant cells in MALDI-TOF analysis. Further, we quantified sphingolipids including SM and Ceramide (Cer) using Multiple Reaction Monitoring (MRM), as they play a vital role in drug resistance. We found that 5-FU resistance in DLD-1/5-FU colorectal cancer cells was mainly associated with SM increase and Cer decrease, which are controlled by acid sphingomyelinase (SMPD1). In addition, reduction of SMPD1 expression was confirmed by LC-MRM-MS analysis and the effect of SMPD1 in drug resistance was assessed by treating DLD-1 cells with siRNA-SMPD1. Furthermore, clinical colorectal cancer data set analysis showed that down-regulation of SMPD1 was associated with resistance to chemotherapy regimens that include 5-FU. Thus, from our study, we propose that SM/Cer and SMPD1 are new potential target molecules for therapeutic strategies to overcome 5-FU resistance.Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality in both men and women 1 . Although there are other drugs for treatment of CRC, 5-Fluorouracil (5-FU) is widely used and is positioned as a first-line chemotherapy. 5-FU was developed as an inhibitor of thymidylate synthase (TS), which results in suppression of thymine synthase, resulting in cell death 2 . The mechanism involves misincorporation of a pyrimidine analogue into RNA and DNA in place of uracil or thymine, respectively 3 . Despite the effectiveness of 5-FU, drug resistance remains a significant limitation. To overcome this drug resistance, many researchers have tried to identify potential genes and proteins involved in mediating 5-FU resistance, using emerging technologies such as microarray profiling 4 and whole genome sequencing 5 . For instance, the alteration of drug influx and efflux by the ABCC5 membrane protein and mutation of the drug target 6 may lead to 5-FU resistance. Furthermore, accumulation of TS protein and elevated activity of deoxyuridine triphosphatase are expected to cause 5-FU resistance www.nature.com/scientificreports www.nature.com/scientificreports/ in CRC. Although various target genes are involved, the detailed 5-FU-resistance mechanism has not been fully elucidated. Therefore, new strategies for therapy and resistance reversal are urgently needed.Various lipidomic approaches have revealed that lipids play key roles in various phenomena in living cells including oncogenesis 7,8 , apoptosis 9 , and drug resistance 10-12 . Alterations in levels of glycerophospholipids (GPs) such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE) h...