Background <p>Fat grafting has been extensively used in plastic surgery practice, yet unstable retention in the recipient site remains a significant clinical challenge. The limited tolerance of injected adipose tissue to ischemia has prompted strategies aiming at timely enhancing the vascularity of the grafted fat. Various modified fat graft preparations have been used, and the mechanically processed tissue stromal vascular fraction (tSVF) derived from fat tissue has garnered considerable interest for enhancing rate of fat graft retention. Further enhancement of the graft retention and quality through supplements to tSVF is worthy of investigation.</p> Methods <p>The arteriovenous (AV) shunt in rats has been used to evaluate tSVF in vivo. We employed this animal model to investigate the regenerative potential of glycolipid Globo H Ceramide (GHCer) added to tSVF isolated from male Lewis rats. Sixty-two rats divided into four groups were studied. Study parameters included gene expression of vascular endothelial growth factor A (VEGFA) and fatty acid binding protein 4 (FABP4), percentages of the CD45⁻CD31⁺ endothelial cell, fat tissue retention and fibrotic changes. In vitro studies on adipose-derived mesenchymal stromal cells (AD-MSCs) included angiogenesis by tube formation assay and adipogenesis.</p> Results <p>The addition of GHCer resulted in superior retention of the tSVF grafts at one-, two-, and eight-week post-grafting (<i>p</i> &lt; 0.05). Elevated expression VEGFA was observed from one week (<i>p</i> &lt; 0.05), followed by FABP4 at two weeks post-grafting in the tSVF + GHCer grafts (<i>p</i> &lt; 0.01). After eight weeks, the numbers of CD45⁻CD31⁺ endothelial cells and adipocytes were significantly increased in the tSVF + GHCer grafts (<i>p</i> &lt; 0.01), while collagen deposition was reduced (<i>p</i> &lt; 0.05). Given that GHCer potentially exerted its effects on tSVF through AD-MSCs within, we performed in vitro studies and demonstrated that GHCer promoted AD-MSC differentiation into neovessels (<i>p</i> &lt; 0.05) and adipocytes (<i>p</i> &lt; 0.001).</p> Conclusions <p>Supplementing GHCer to tSVF effectively reduced fat reabsorption and fibrotic changes of the grafts, while enhancing angiogenesis and adipogenesis, potentially through facilitating AD-MSC differentiation within tSVF. These findings support the potential clinical application of GHCer to enhance the stability and long-term outcomes of fat grafting procedures.</p> Trial registration <p>Not applicable.</p> Clinical trial number <p>Not applicable.</p>