The senescence of mesenchymal stem cells (MSCs) impairs their regenerative capacity to maintain tissue homeostasis. Numerous studies are focusing on the interventions and mechanisms to attenuate the senescence of MSCs. C-phycocyanin (C-PC) is reported to have multiple functions such as antitumor, antioxidation, anti-inflammation and anti-aging roles, but there is little research about the effects of C-PC on the senescence of MSCs. Here we investigated the roles and mechanism of C-PC on MSCs senescence. In vitro results showed that C-PC could reduce senescence, enhance proliferation, promote the adipogenic and osteogenic differentiation in senescent MSCs induced by oxidative stress. In vivo D-Galactose (D-Gal) induced rats aging models showed C-PC also increased the viability and differentiation of intrinsic senescent bone marrow derived MSCs (BMSCs). Furthermore, C-PC also decreased the levels of oxidative stress markers ROS or MDA, elevated the SOD activity, and increased the anti-inflammatory factors. Proteomic chip analysis showed that C-PC interacted with ZDHHC5, and their interaction was verified by pull down assay. Overexpression of ZDHHC5 aggravated the senescence of MSCs and greatly lessened the beneficial effects of C-PC on senescence. In addition, we found ZDHHC5 regulated autophagy by altering LC3, Beclin1 and PI3K/AKT/mTOR pathway. In summary, our data indicated that C-PC ameliorates the senescence of MSCs through zinc finger Asp-His-His-Cys (DHHC) domain-containing protein 5 (ZDHHC5) mediated autophagy via PI3K/AKT/mTOR pathway. The present study uncovered the key role of autophagy in MSCs senescence and PI3K/AKT/mTOR pathway may be a potential target for anti-senescence studies of MSCs.