Imatinib mesylate (IM) is highly effective in the treatment of gastrointestinal stromal tumors (GISTs). However, the most of GISTs patients develop secondary drug resistance after 1-3 years of IM treatment. The aim of this study was to explore the IM-resistance mechanism via the multi-scope combined with plasma concentration of IM, genetic polymorphisms and plasma sensitive metabolites.This study included a total of 40 GISTs patients who had been regularly treated and not treated with IM. The plasma samples were divided into three experiments, containing therapeutic drug monitoring (TDM), OCT1 genetic polymorphisms and non-targeted metabolomics. According to the data of above three experiments, the IM-resistant cell line, GIST-T1/IMR cells, was constructed for verification the IM-resistance mechanism.The results of non-targeted metabolomics analysis suggested that the sphingophospholipid metabolic pathway including the SPK1/S1P axis was inferred in IM-insensitive patients with GISTs. A GIST cell line (GIST-T1) was immediately induced as an IM resistance cell model (GIST-T1/IMR) and we found that blocking the signal pathway of SPK1/S1P in the GIST-T1/IMR could sensitize treatment of IM and reverse the IM-resistance.Our findings suggest that IM secondary resistance is associated with the elevation of S1P, and blockage the signaling pathway of SPK1/S1P warrants evaluation as a potential therapeutic strategy in IM-resistant GISTs. The design of this study from blood management, group information collection, IM plasma concentration with different elements, identification of sphingolipid metabolism and lastly verification the function of SPK1/S1P in the IM-resistance GISTs cells.© 2022. The Author(s) under exclusive licence to The International Gastric Cancer Association and The Japanese Gastric Cancer Association.