Adipose-derived stem cell-conditioned medium (ASC-CM) is promising for cardiac repair via paracrine mechanisms. However, variability in efficacy limits its clinical translation. We investigated whether preconditioning human ASC with butyrate (ASC-BA-CM) enhanced its paracrine potency and improved in vitro and in vivo outcomes. RNA-sequencing of human ASCs treated with butyrate was performed to characterize transcriptomic changes. CM was collected and analyzed via cytokine/chemokine arrays. Wound healing assays using human umbilical vein endothelial cells (HUVECs), with and without THP-1 macrophage co-culture, were performed to evaluate endothelial repair and its correlations with secreted factors. In vivo angiogenesis was assessed using a sponge implantation model, and myocardial perfusion was measured in a rat myocardial infarction model using single-photon emission computed tomography/computed tomography (SPECT/CT) thallium-201 imaging. Butyrate preconditioning upregulated angiogenesis- and immune-related genes, including CXCL8, SOD2, and TGM2. It increased IL-10, CXCL5, and MMP-1 secretion. In vitro, BA-preconditioned ASC-CM enhanced HUVEC wound closure, which was improved by co-culture with THP-1 macrophages and negatively correlated with TGFb3 and TIMP-2 levels. In vivo, ASC-BA-CM promoted vascularization and macrophage accumulation in sponges and significantly improved myocardial perfusion by approximately 32 % compared with controls. Butyrate preconditioning enhanced the paracrine activity of ASC-CM and was associated with improved myocardial perfusion in a rat model. These findings suggest that butyrate may augment the ASC secretome function. Potential mechanisms such as endothelial repair, angiogenesis, and immune modulation remain hypothetical and require further validation in future studies.