HMGb1 is a nuclear protein playing a role in DNA architecture and transcription. This protein has also been shown to function as a cytokine and to stimulate keratinocyte scratch wound healing. Due to the importance of finding new wound healing molecules, we have studied the effects of HMGb1 on fibroblasts, another major skin cell type, using the NIH 3T3 line. HMGb1 expression in these cells was assessed by Western blot, while its nuclear localization was pointed out by confocal immunofluorescence. HMGb1-induced cell proliferation with a maximum at a concentration of 10 nM, and such a dose also stimulated cell migration and scratch wound healing. Western blot analysis showed that HMGb1 activates ERK1/2, while the use of an anti-RAGE receptor-blocking antibody and of the selective MEK1/2 inhibitor PD98059 blocked ERK1/2 activation and wound healing responses to HMGb1. Taken together data show that HMGb1 promotes 3T3 fibroblast wound healing by inducing cell proliferation and migration, and that this occurs through the activation of the RAGE/MEK/ERK pathway. In conclusion, HMGb1 seems a good candidate for the development of medical treatments to be used on chronic or severe wounds.