Background and Aim: Angiogenesis and anti-apoptosis play crucial roles in ischemic stroke recovery. The mesenchymal stem cell (MSC) secretome, rich in bioactive molecules, presents a promising therapeutic avenue. However, optimizing the culture conditions to enhance the expression of angiogenic markers remains a challenge. This study examines the effect of hypoxic preconditioning on the expression of vascular endothelial growth factor (VEGF), monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinase-2 (MMP-2), and microRNA (miRNA-128) in the secretome of umbilical cord-derived MSCs (UC-MSCs) from Macaca fascicularis. Materials and Methods: UC-MSCs were cultured under normoxic (21% O2) and hypoxic conditions (1%, 3%, and 5% O2) for 48 h. The secretome was isolated, and reverse transcription-quantitative polymerase chain reaction was used to quantify the expression of VEGF, MCP-1, MMP-2, and miRNA-128. Expression levels were normalized to housekeeping genes and analyzed using statistical methods to determine significant differences among groups. Results: Hypoxic preconditioning significantly upregulated VEGF (1% O2), MCP-1 (5% O2), and miRNA-128 (5% O2) expression compared to normoxic conditions. Conversely, MMP-2 expression was highest in normoxic conditions and downregulated under hypoxia. In addition, miRNA-128 was found to be predominantly secreted into the extracellular space under hypoxic conditions rather than retained within cells. Conclusion: Hypoxic preconditioning effectively modulates the expression of key angiogenesis and anti-apoptotic markers in UC-MSCs. The study highlights the importance of optimizing oxygen levels to enhance the therapeutic potential of MSC-derived secretomes for ischemic stroke treatment. Future research should focus on in vivo validation and clinical translation of these findings. © 2025 Veterinary World. All rights reserved.