The major facilitator superfamily domain containing 8 (MFSD8) belongs to an orphan transporter protein expressed in a wide range of tissues. Nevertheless, the specific role of MFSD8 in human health and disease remains unknown. This study aimed to evaluate the role of MFSD8 protein on metabolic function using untargeted metabolomics analysis in human umbilical vein endothelial cells (HUVECs). HUVECs overexpressing MFSD8 were subjected to metabolomics analysis to evaluate changes in endogenous small molecules using LC-MS/MS analysis. In the positive scan mode, 634 metabolites from 1583 compounds were identified. In the negative scan mode, 169 metabolites from 405 compounds were identified. According to the established criteria for identifying differential metabolites, 96 metabolites exhibited significant differences between the MFSD8 and Vector groups. Among them, 62 metabolites were found to be up-regulated, whereas 34 metabolites were classified as down-regulated. Bioinformatics pipeline analysis revealed three common metabolic pathways, including arginine biosynthesis, beta-alanine metabolism, and pyrimidine metabolism, were found under the positive and negative scan modes. The semi-quantitative analysis was conducted on the differential metabolites, revealing that overexpression of MFSD8 resulted in increased levels of L-citrulline, L-aspartic acid, ornithine, N-acetyl-l-aspartic acid, L-histidine, beta-alanine metabolites and exhibited decreased levels of cytidine. The findings of our study indicated that MFSD8 had the most significant role in arginine biosynthesis, beta-alanine metabolism, and pyrimidine metabolism pathways within endothelial cells. The metabolomics data provide new insights into studying potential features of MFSD8 protein in the future.