Background: Identification of a safe and convenient substance capable of preventing androgenetic alopecia and promoting the growth of hair on the scalp remains problematic. Objectives: This study was designed to investigate the effectiveness of a newly developed topical agent containing human umbilical cord blood mesenchymal stem cell (hUCB-MSC)-derived conditioned media on promotion of hair growth. Methods: This was a double-blinded, placebo-controlled clinical study of outcomes in 30 patients with patterned hair loss treated with a topical agent containing hUCB-MSC-derived conditioned media. The efficacy of the treatment was determined using phototrichograms, which were used to evaluate the density, diameter, and growth rate of hair at baseline after treatment for 4, 8 and 16 weeks. Results: The hair density of the group treated with the topical agent was significantly increased from 96.7 to 110.1 hairs/cm². The hair thickness of the topical agent group also increased from 0.074 to 0.094 mm. In addition, the hair growth rate in the test group increased from 0.262 to 0.312 mm/day. No severe adverse reactions were reported. Conclusion: A topical agent containing hUCB-MSC- derived conditioned media described in this study may be a new effective treatment option in patients with androgenetic alopecia.