Abstract Aim TPD52 (tumor protein D52) and TPD52L2 (tumor protein D52-like 2), members of the TPD52 gene family, have been implicated in multiple malignancies. However, their roles in gastric cancer (GC) remain elusive. Herein, we integrated multiomics analyses and experimental validation to elucidate their prognostic and functional significance in GC. Methods Utilizing The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and tissue microarray datasets, we analyzed TPD52/TPD52L2 expression patterns in patients with GC. Survival analysis, Cox regression, and nomogram construction were performed to assess prognostic value. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes functional enrichment analysis and immune infiltration evaluation (Cell-type Identification By Estimating Relative Subsets Of RNA Transcripts‌/Estimation of STromal and Immune cells in MAlignant Tumour tissues using Expression data) (CIBERSORTx/ESTIMATE) were conducted to explore the molecular mechanisms involved. In vitro experiments (cell proliferation, migration, invasion, and apoptosis assays) were performed via lentivirus-mediated gene knockdown in gastric cancer cell lines AGS and MKN45 cells. Results TPD52 and TPD52L2 were significantly overexpressed in GC tissues compared with their normal counterparts. Elevated TPD52L2 expression was significantly associated with advanced Tumor, Node, Metastasis (TNM) stage and independently predicted reduced overall survival according to multivariate Cox regression. Multivariate analysis identified TPD52L2 as an independent prognostic factor. Diagnostic Receiver Operating Characteristic (ROC) curves yielded area under the curve values of 0.813 (TPD52) and 0.807 (TPD52L2). The results of functional experiments suggested that TPD52/TPD52L2 knockdown inhibited proliferation, migration, G0/G1 arrest, and induced apoptosis. Mechanistically, TPD52/TPD52L2 silencing suppressed PI3K/Akt serine/threonine kinase (AKT)/mammalian target of rapamycin (mTOR) signaling and epithelial–mesenchymal transition marker expression. Conclusion TPD52 and TPD52L2 are promising prognostic biomarkers in GC, with TPD52L2 exhibiting greater clinical relevance. Targeting these proteins may disrupt oncogenic signaling pathways and enhance immunotherapy efficacy, warranting further investigation in clinical trials.