Hydroxysafflor yellow A (HSYA) is the main bioactive component of safflower and has been reported to have significant health-promoting abilities. However, the regulation of HSYA on different types of skeletal myofibers is largely unknown. Here, in vitro experiments found that the water extract of safflower could significantly increase MyHC I, MB and Tnni1 mRNA expression while downregulating MyHC IIb mRNA expression. Furthermore, HSYA triggered fast-to-slow fiber-type switching and increased gene expression related to mitochondrial biosynthesis both in vitro and in vivo. Autodock analyses proved that FoxO1 is a potential target of HSYA, and qRT-PCR and western blotting further showed that HSYA significantly promoted the activation of the FoxO1 signaling pathway. Additionally, the levels of PGC1α, downstream of FoxO1, also significantly increased after HSYA treatment. Together, our findings suggested that HSYA triggered a fast-to-slow myofiber-type shift through the FoxO1 signaling pathway.