Inonotus obliquus, an edible and medicinal fungus, has proven to be beneficial for relieving body aging. Previous research has shown that I. obliquus polysaccharide (IOP) can effectively reduce oxidative stress and alleviate photoaging in UVB-treated HaCaT cells. However, the underlying mechanism is still unclear. This study aimed to investigate the mechanism through which IOP mitigates skin photodamage by modulating mitochondrial autophagy through both in vivo and in vitro experiments. The results indicated that IOP alleviated the thickening of the stratum corneum and inflammatory infiltration caused by UVB radiation. Moreover, IOP effectively inhibited the mRNA expression of matrix metalloproteinase-1, 3 (MMP1 and MMP3) to prevent collagen breakdown and decreased the expression of aging-related proteins (P53 and P21) and inflammatory factors (tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in UVB-induced mice. Both in vitro and in vivo studies demonstrated that IOP increased Sirtuin3 (Sirt3) expression, activated the Foxo3a/PINK1-Parkin pathway and enhanced mitochondrial autophagy to reduce photodamage. Additionally, IOP could regulate the gut microbes in photodamaged mice and increase the relative abundance of Clostridium_XlVa, Desulfovibrio, and Barnesiella, which are important for protecting the intestinal barrier and alleviating intestinal dysfunction. These results provide a theoretical foundation for the use of IOP as a natural component to reduce skin photodamage.