Objectives: Application of the picosecond laser in the field of dermatology has expanded from tattoo removal to skin rejuvenation on a clinical basis. Although various mechanisms of pigment removal have been elucidated, the molecular changes associated with skin rejuvenation have yet to be identified. The aim of this study was to explore the theoretical basis and to evaluate the efficacy of skin rejuvenation using a 1064-nm fractional picosecond laser in a mouse model. Methods: We conducted an in vivo study using a fractional picosecond laser on the skin of old and young female hairless mice and performed topographical, histological, micro-, and electron microscopic assessments. Results: The topography of the skin surface was enhanced and showed increased dermal thickness on histological examination. Electron microscopy revealed disarranged collagen bundles with microspaces and vascular leakage in the upper dermis. Levels of collagen synthesis markers and various inflammatory cytokines, such as procollagens, interleukin-1β, tumor necrosis factor-α, and heat shock proteins, were elevated in the laser-treated skin. Conclusions: This study provides a possible mechanism for the skin rejuvenation effect of fractional picosecond laser that has been reported previously in clinical observations. Based on our findings, the fractional picosecond laser could be widely applied in clinical settings where dermal regeneration and promotion of skin rejuvenation is required.