Resistance to 5-FU reduces its clinical efficacy for the treatment of colorectal cancer. Sphingosine-1-phosphate receptor 2 (S1PR2) has emerged as a potential target to reverse 5-FU-resistance by inhibiting the expression of dihydropyrimidine dehydrogenase (DPD). In this study, 38 novel S1PR2 antagonists based on aryl urea structure were designed and synthesized, and the structure-activity relationship was investigated based on the S1PR2 binding assay. Representative compound 43 potently interacts with S1PR2 with a KD value of 0.73 nM. It displays potent 5-FU resensitizing activity in multiple 5-FU-resistant tumor cell lines, particularly in SW620/5-FU (EC50 = 1.99 ± 0.03 μM) but shows no cytotoxicity in the normal colon cell line NCM460 up to 1000 μM. Moreover, 43 significantly enhances the antitumor efficacy of 5-FU in the SW620/5-FU animal model. These data suggest that 43 could be a novel lead compound for developing a 5-FU resensitizing agent.