Sphingosine-1-phosphate receptor-1 (S1PR1), a G protein-coupled receptor, has been reported to be involved in lymphangiogenesis. Degradations of extracellular matrix (ECM) are recognized as dynamic modulators in regulating the formation of new lymphatic vessels. However, little research has studied the ECM on S1PR1 in the regulation of lymphatic endothelial cells (LECs) in tumor lymphangiogenesis. Here we attempt to investigate hyaluronan fragments abundant in tumor microenvironment (TME) on S1PR1 in new lymphatic vessel formation. First, we verified that low molecular weight hyaluronan (LMW-HA) derived from tumor cells could promote LECs migration and capillary-like tube formation. Then, we demonstrated that S1PR1 on LECs underwent internalization into the endoplasmic reticulum in response to LMW-HA treatments. Notably, the S1PR1 endocytosis could upregulate lymphangiogenesis. Next, we found that the ablation of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) could attenuate the S1PR1 endocytosis, implying a novel role of LMW-HA/LYVE-1 in the S1PR1 cycling pathway. Furthermore, we identified that LMW-HA/LYVE-1 interaction could activate Src kinase which in turn upregulates S1PR1 tyrosine phosphorylation, resulting in S1PR1 endocytosis. Collectively, our findings suggested that hyaluronan fragments in TME could induce S1PR1 internalization in LECs, leading to lymphangiogenesis promotion.