Background <p>The human umbilical cord (hUC)–mesenchymal stem cell (MSC) secretome (SCT) is a cell-free therapy that may emerge as a novel therapeutic strategy for hair loss prevention. Here, we aimed to elucidate the underlying mechanisms through which SCT regulates hair growth and cycle transition.</p> Methods <p>Using C57BL/6 mice, ex vivo follicles, and cell experiments, we studied the effects and mechanisms of SCT on hair growth and cycling using untargeted metabolomics and phosphoproteomics. A three-month double-blind clinical study was conducted to validate the effects of SCT on human hair.</p> Results <p>SCT promotes the telogen-to-anagen transition, hair thickening, and elongation of the vibrissae in mice; regulates dermal papilla cells and hair matrix cells through cysteine and methionine metabolism; and stimulates methylthioadenosine synthesis in hair matrix cells by activating the PI3K/AKT/mTOR signaling pathway. Clinical studies demonstrated that SCT increased human hair density and average hair diameter. Scalp physiological tests and subjective feedback indicated no related adverse reactions on the scalp or hair.</p> Conclusions <p>SCT promoted hair growth, thickening, and the hair follicle cycle via the PI3K/AKT/mTOR signaling pathway. This research provides a basis for the application of cell-free alternatives in hair care and hair loss prevention.</p> Graphical Abstract <p></p>