The aim of the present study was to investigate the expression, function and underlying molecular mechanism of the long non‑coding (lnc) RNA RP1‑163G9.1 in patients with gastric adenocarcinoma (GA). The expression levels of lncRNA RP1‑163G9.1 were determined in 112 paired clinical GA tissues by reverse transcription‑quantitative polymerase chain reaction analysis. Subsequently, the potential clinical values of lncRNA RP1‑163G9.1 were analyzed with statistical methods. Additionally, the function of lncRNA RP1‑163G9.1 was explored at the cellular level using the Cell Counting Kit‑8 proliferation assay, Transwell experiments, fluorescence in situ hybridization (FISH), colony formation assay and flow cytometry. Furthermore, the function of lncRNA RP1‑163G9.1 was assessed in vivo using subcutaneous tumorigenesis experiments in nude mice. lncRNA RP1‑163G9.1 expression in GA tissues and cells was significantly decreased when compared with that in control gastric tissues (P<0.001) or gastric epithelial cells GES‑1 (P<0.05). This finding was associated with the depth of invasion (P=0.001), lymph node metastasis (P=0.009), tumor size (P=0.037) and immunocytochemistry marker Ki‑67 (P=0.010). FISH detection demonstrated that lncRNA RP1‑163G9.1 was primarily located in the cytoplasm. Notably, overexpression of lncRNA RP1‑163G9.1 significantly decreased cell proliferation (P<0.01), colony formation (P<0.01), invasion (P<0.01) and the number of cells at the S‑phase of the cell cycle (P<0.05); However, it did not exert a significant effect on apoptosis (P>0.05). Furthermore, tumor formation experiments revealed that overexpression of lncRNA RP1‑163G9.1 inhibited cancer cell proliferation in nude mice. The present research indicated that low expression of lncRNA RP1‑163G9.1 may be associated with enhanced tumor proliferation and invasion in GA.