BackgroundChronic refractory wounds easily relapse and seriously affect the patients’ quality of life. Previous studies have shown that stromal vascular fraction gel (SVF-gel) significantly promotes the early healing of chronic wounds; however, the mechanisms of SVF-gel function per se remain unclear, and a long-term follow-up is lacking. This study aims to explore the mechanisms of SVF-gel promoting the healing of chronic wounds and follow up the long-term efficacy of SVF-gel.MethodsAutologous SVF-gel transplantation was performed in 20 patients with chronic wounds (from March 2016 to September 2019), and the size of the wound before and after SVF-gel transplantation was observed. The conditioned medium (CM) was harvested from SVF-gel under serum-free, serum-deprivation and 10% fetal bovine serum (FBS) microenvironment in vitro, respectively. The concentration of the growth factors in the two kinds of gel-CM was tested, and their effects on the proliferation and migration of human dermal fibroblasts (HDFs) were detected.ResultsAll patients had 100% wound closure eventually, and the average time to complete closure was 28.3 ± 9.7 days. The time of follow-up ranged from 2 to 6 years, and there was no wound recurrence. Interestingly, the concentrations of epidermal growth factor and transforming growth factorβ1 of the CM were higher in serum-free and serum-deprivation condition than in 10% FBS microenvironment (p< 0.05). Correspondingly, the proliferation and migration ability of HDFs treated with gel-CM from serum-free condition were stronger than those treated with gel-CM from serum-deprivation (2% FBS) or 10% FBS microenvironment (p< 0.05).ConclusionThese results indicate that it is safe, effective, and lasting in effect to treat chronic wounds with SVF-gel and mechanisms of action that include secreting various cytokines and promoting cell proliferation and migration ability.Trial registration: Chinese Clinical Trail Registry, ChiCTR2000034624. Registered 12 July 2020—Retrospectively registered,http://www.chictr.org.cn/showproj.aspx?proj=56058Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-023-03389-2.