BACKGROUND: Gastric cancer (GC), a prevalent aggressive form of tumor, imposes a significant burden in terms of morbidity and mortality. Prolyl 4-hydroxylase, alpha polypeptide I (P4HA1), a key enzyme in collagen synthesis, comprises two identical alpha subunits and two beta subunits. Studies on the expression and impact of P4HA1 in GC cells are limited. METHODS: The expression and prognosis of P4HA1 in GC were analyzed using bioinformatics. To confirm the P4HA1 level in GC tissues and cells, Western blot (WB) and RT-qPCR experiments were conducted. The signaling pathways related to P4HA1 in GC were examined using the DAVID database. Moreover, the expression of P4HA1 was downregulated by transfecting GC cell lines (HGC-27 and SGC-7901) with siRNA technology. Furthermore, GC proliferation, migration, and invasion were detected via plate cloning, CCK-8, and Transwell assays. The epithelial-mesenchymal transition (EMT) genes (E-cadherin, N-cadherin, Vimentin) and the stemness marker CD44 protein expression in GC cells were detected using WB. The sphere-forming ability of GC cells was analyzed using a sphere-forming assay to determine the effect of P4HA1. RESULTS: Bioinformatics and experimental analyses demonstrated that P4HA1 expression was extensively detected in GC tissues and cells, and strongly related to a poor prognosis for GC. In vitro studies demonstrated that P4HA1 suppression hindered the proliferation, migration, and invasion of GC cells and suppressed EMT characteristics. Both sphere-forming and WB assays revealed that the sphere-forming potential of GC cells and the level of CD44 protein decreased after knocking down the expression of P4HA1, indicating that suppression of P4HA1 could inhibit the stemness of GC cells. CONCLUSION: The study concluded that P4HA1 has the potential to be expressed substantially in GC tissues and cells and is capable of enhancing the proliferation, metastasis, and stemness of GC.