Background <p>Phosphatidylserine synthase 1 (<i>PTDSS1</i>) inhibitor has been developed and shows promising anti-tumor effect, but existing studies about <i>PTDSS1</i> remain limited in cancer.</p> Methods <p>This study preliminarily analyzed the differential expression, prognostic significance, copy number variation, methylation level, co-expression network and immune correlation of <i>PTDSS1</i> in pan-cancer. Furthermore, we collected tumor samples and corresponding clinical information of 74 hepatocellular carcinoma (HCC) patients from the First Hospital of Guangxi Medical University (Guangxi cohort) to validate <i>PTDSS1</i> expression and prognostic value. In vitro experiments were performed to investigate the effect of <i>PTDSS1</i> knockdown on the malignant phenotypes of HCC cell lines.</p> Results <p>This study identified that <i>PTDSS1</i> was significantly upregulated across most cancer types and associated with poor prognosis of seven cancer types. Copy number amplification and hypermethylation of <i>PTDSS1</i> may drive its upregulation. Furthermore, <i>PTDSS1</i> exhibited a positive correlation with macrophage in various cancers, co-expression network and functional enrichment analysis revealed that <i>PTDSS1</i> may involve in cell division, cell cycle, DNA repair, cell proliferation, cell migration, cell growth, immune response and WNT signaling pathway. Regarding HCC, the differential expression and prognostic significance of <i>PTDSS1</i> were verified by Guangxi cohort, single cell sequencing analysis revealed <i>PTDSS1</i> located in tumor cell and macrophages. In vitro experiments demonstrated that <i>PTDSS1</i> knockdown suppressed the proliferation, migration, invasion of HCC cells and promoted macrophage repolarization towards the M1 phenotype.</p> Conclusions <p><i>PTDSS1</i> serves as a diagnostic and prognostic biomarker associated with macrophage infiltration, and may be a promising therapeutic target.</p>