Background: ACSS3 (acyl-CoA synthetase short-chain family member 3) is found in numerous tissues and is linked to tumor cell type development and metastasis. Methods: We conducted a comprehensive pan-cancer analysis of ACSS3. The TCGA (Cancer Genome Atlas), CPTAC (Clinical Proteomic Tumor Analysis Consortium), and HPA databases were used to ascertain the connection between ACSS3 and various types of tumors. Genes in the TCGA database would be identified using cBioPortal queries, and their transcriptome expression would then be verified using GEO data. ACSS3 expression and cellular localization in various tumor tissues of most cancer types were analyzed using single-cell sequencing data from the TISCH database. According to HPA and CPTAC databases, we analyzed and evaluated protein expression levels. Predictive analysis based on precise survival data of ACSS3 expression levels for 26 cancer types predicted using the TCGA database. Furthermore, we investigated the relationship between ACSS3 and immune microenvironments in different tumor tissues using the TIMER and TISCH databases. CellMiner, GDSC, and CTRP data would clarify the relationship between ACSS3 and drug resistance and explore the chemicals that affect ACSS3 expression. The final part of our study explored and validated the role ACSS3 played in glioma proliferation, migration, and invasion. Results: ACSS3 is differentially expressed in various tumors and exhibits early diagnostic value. ACSS3 expression is associated with clinical features, and high ACSS expression anticipates a worse prognosis in multiple tumors and may impact drug sensitivity. The changes in the immunosuppressive microenvironment of gliomas are closely related to the upregulation of ACSS3. Conclusions: ACSS3 is a novel biomarker for forecasting different human cancer prognoses, as it can influence the biological process by modulating the immune microenvironment. ACSS3 is a critical prognostic factor for glioma and is related to its proliferation, migration, and invasion.