Background and Purpose: Dihydropyrimidine dehydrogenase (DPD) is a major determinant of 5-FU resistance in cancers. DPD catalyzes 5-FU into FBAL (α-fluoro-β-alanine) to lower intracellular 5-FU level. We aimed to investigate mechanism and clinical significance of FBAL-stimulated sphingosine 1-phosphate receptor 2 (S1PR2) in upregulation of DPD in colonic cancer. Experimental Approach: Cancer cells transfected or silenced S1PR2 were exposed to FBAL for analyzed S1PR2 and DPD levels. Luciferase reporter assay analyzed S1PR2-activated TWIST1 binding to DPYD promoter. Co-IP assay analyzed TWIST1 interaction with JMJD3-RNA Pol II complex binding to DPYD promoter. HT-29sh-S1PR2 or SW480TgS1PR2 cells xenografted nude mice were used to evaluate clinical significance of S1PR2-upregulated tumoral DPD. Key Results: Activation of S1PR2 upregulated DPD in colonic cancer cells and human fresh cancer specimens. FBAL was first time identified as an etiological stimulator of S1PR2 activation. The FBAL-stimulated S1PR2 increased TWIST1 binding to DPYD promoter and interacting with JMJD3-RNA Pol II complex, enhancing H3K27me3-enriched DPYD transcription elongation. Transfection of S1PR2 in SW480TgS1PR2 xenograft contributed 5-FU resistance by 45.14%, and silence of S1PR2 improved 5-FU sensitivity by 62.12% in HT-29sh-S1PR2 xenograft. S1PR2 inhibitor JTE013 prevented the FBAL-stimulated S1PR2’s effects in upregulating DPD. Cancer cells with high S1PR2 are more resistant to 5-FU, strongly suggesting clinical significance that combination use of S1PR2 inhibitors may be the appropriate 5-FU-based regimens of colonic cancers. Conclusions and Implications: The FBAL-stimulated S1PR2 increased TWIST1 binding to DPYD promoter and interacting with JMJD3-RNA Pol II, enhancing the H3K27me3-enriched DPYD transcription elongation.