Abstract: Background　sphingosine-1-phosphate(S1P), an important lipid mediator, is involved in the regulation of multiple pathophysiological processes. Among its receptors, S1PR3 plays a significant role in the development and progression of various tumors; however, its specific function in hepatocellular carcinoma (HCC) remains unclear.Objective　To investigate the effect of sphingosine-1-phosphate receptor 3(S1PR3) on the malignant biological behavior of HCC and to preliminarily elucidate its mechanism of action.Methods　Using DMSO treatment as a control, the S1PR3-specific inhibitor TY-52156 was applied to human HCC cell lines Huh7 and HepG2. Cell proliferation was assessed by CCK-8 assay; cell migration and invasion abilities were evaluated using Transwell assays; differentially expressed genes and potential signaling pathways were screened through Transcriptome sequencing; key molecule expression was verified by Western blot; a liver cancer organoid model was established, and cell viability after inhibitor treatment was measured by the CellTiter-Glo assay.Results　Compared to the control group, both CCK-8 and Transwell assays demonstrated that TY-52156 inhibited the proliferation, migration, and invasion of Huh7 and HepG2 cells, with statistical significance (P＜0.05). Transcriptome sequencing and subsequent analysis suggested that argininosuccinate synthetase 1 (ASS1) may function as a downstream effector of S1PR3 involved in the regulatory process; Western blot results showed that inhibition of S1PR3 upregulated ASS1 expression (P＜0.05). In the organoid model, the CellTiter-Glo assay indicated that treatment with TY-52156 induced tumor organoid death compared to the control group (P＜0.001).Conclusion　Inhibition of S1PR3 impairs the proliferation, migration, and invasion abilities of HCC cell lines. As a downstream differentially expressed molecule of S1PR3, ASS1 expression is negatively regulated by S1PR3. S1PR3 inhibitors effectively kill HCC organoids.