Objective: To study the protective effect of adipose-derived stem cell conditioned medium against ultraviolet radiation B (UVB)-induced photoaging and replicative senescence in human dermal fibroblasts (HDFs). Methods: HDFs were cultured and passaged in vitro, and treated with ADSC-CM after being irradiated once with UVB. The mRNA expression of CollagenⅠ(Col1), CollagenⅢ(Col3) and Elastin were detected by real-time PCR to define the anti-aging effects of ADSC-CM. The protein expressions of phosphorylated c-Jun N-terminal kinase (p-JNK) and p-p38 were detected by Western blot. Results: ①Both successive passage and UVB irradiation reduced the expression of Col1, Col3 and Elastin in HDFs.②ADSC-CM inhibited the reduction of Col1, Col3 and Elastin protein expressions induced by successive passage and UVB. ③ADSC-CM activated p38 and JNK pathway. Downregulation of p38 MAPK by SB203580 decreased the mRNA expression of Col1, Col3, and Elastin. Inhibiting JNK by SP600125 did not induce significant ECM changes. Conclusions Both intrinsic and extrinsic stimuli can decrease the expression of collagen and elastin, common markers of skin aging in HDFs, and ADSC-CM can attenuate the above decreasing and promoting the expression of ECM by p38 signaling pathway.