BACKGROUNDS: Gastric cancer-associated mesenchymal stem cells (GC-MSCs) as integral components of the tumor microenvironment potentiate gastric cancer growth and metastasis. SALL4 is aberrantly upregulated in gastric cancer and pivotal for malignant progression. Whether GC-MSCs is responsible for SALL4 upregulation and the underlying mechanisms remains elusive. METHODS: Cancer growth and metastasis capacities were assessed by cell colony formation assay, transwell assay, and epithelial-mesenchymal transition protein detection in vitro as well as subcutaneous xenograft and peritoneal metastasis models in vivo. SALL4 was measured by qPCR, western blot and immunohistochemistry staining. Gain- and loss-functional analysis were performed for miRNA and target gene. β-catenin signaling was assessed by immunofluorescence staining and Top/FopFlash luciferase assay. Transcriptional regulation was conducted using chemicals, luciferase reporter and ChIP assay. Clinical tissues and TCGA-STAD database were included for expression profile, correlation and clinical relevance analysis. RESULTS: GC-MSCs promoted gastric cancer growth and metastasis along with elevation of SALL4 and miR-4669 in cancer cells and tissues. Overexpression of miR-4669 mimicked GC-MSC effects, while miR-4669 knockdown eliminated their oncogenic roles. TIMP3 was identified as a target of miR-4669 and mediated its functions. TIMP3 overexpression counteracted GC-MSC-induced cancer progression and SALL4 expression. GC-MSCs activated SALL4 transcription through the miR-4669/TIMP3/β-catenin pathway. The regulatory axis was aberrantly expressed in gastric cancer tissues, correlated with each other in certain cancer tissues and associated with lymph node metastasis. CONCLUSIONS: GC-MSCs transcriptionally upregulate SALL4 to facilitate gastric cancer cell growth and metastasis via miR-4669/TIMP3/β-catenin pathway, highlighting the crucial role of GC-MSCs in the aberrant upregulation of SALL4.