BACKGROUND Klotho protein plays a pivotal role in aging regulation. However, it is unclear whether klotho is expressed in human hair follicles and is correlated with hair growth. OBJECTIVE The purpose of this study was to determine the expression pattern and role of klotho in human hair follicles. METHODS We examined the klotho expression patterns in human hair follicles from young and aged donors. Furthermore, we examined the functional roles of klotho on human hair growth using klotho siRNA and klotho recombinant protein. RESULTS Interestingly, klotho was expressed in human hair follicles at both gene and protein levels. In hair follicles, prominent klotho expression was mainly observed in the outermost regions of the outer root sheath and hair bulb matrix cells. Quantification of klotho protein expression in young and aged donors showed that klotho expression decreased with aging. In human hair follicle organ culture, klotho silencing promoted premature catagen induction and inhibited human hair growth. Otherwise, klotho protein prolonged human hair growth. CONCLUSIONS These results indicate that klotho might be an important regulatory factor for human hair growth and hair cycle change.