Primary retroperitoneal liposarcoma (RLPS) is a rare heterogeneous tumor occurring within retroperitoneal space, and its overall survival has not improved much in the past few decades. Based on a small-sample clinical practice at our center, patients with RLPS can greatly benefit from anlotinib and eribulin combination. In this study, we investigated the combinational effect of anlotinib and eribulin on RLPS. In vitro experiments revealed that a low dose of anlotinib significantly enhances the cytotoxic effects of eribulin, leading to a remarkable suppression of RLPS cell proliferation, viability, colony formation, migration, and cell-cycle progression compared to individual drug treatments. At the organoid level, the combination treatment causes the spheroids in Matrigel to disintegrate earlier than the single-drug group. In vivo, RLPS patient-derived xenograft (PDX) models demonstrated that the combination of these two drugs can obviously exert a safe and effective anti-tumor effect. Through transcriptome analysis, we uncovered and validated that the synergistic effect mainly is induced by the endoplasmic reticulum stress (ERS) pathway both in vitro and in vivo. Further analyses indicate that anlotinib plus eribulin treatment results in micro-vessel density and PD-L1 expression alterations, suggesting a potential impact on the tumor microenvironment. This study extensively explored the combination regimen at multiple levels and its underlying molecular mechanism in RLPS, thus providing a foundation for translational medicine research.