The purpose of the current study was to investigate the expression and effects of ornithine decarboxylase (ODC) in human hair follicles. We examined the expression of ODC in human hair follicles and its expression change in organ cultured human hair follicles. Furthermore, we examined the effects of ODC on follicular keratinocyte (FKC) growth and proliferation/apoptosis-related molecular expression using α-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC and small interfering RNA (siRNA) technique. Human hair follicles expressed ODC at mRNA and protein levels. ODC immunoreactivity can be detected in the epithelium of human anagen hair follicles. During the transformation from anagen to catagen, ODC expression appeared to be down-regulated. DFMO inhibited the proliferation of FKC dose-dependently. The levels of P-Erk, P-Akt, and Bcl-2 decreased while Bax increased after treatment of DFMO. Gene transfection of ODC siRNA markedly down-regulated the expression of ODC, resulting in the suppression of FKC proliferation. Moreover, DFMO significantly inhibited human hair growth and promoted apoptosis in organ cultured human follicles. Altogether, these results indicate that ODC might be an important regulatory enzyme for human hair growth and hair cycle change.