The mechanism of the regulation on photosynthesis after spaceflight has not been fully understood. To learn more information about this, we conducted a series of experiments of photosystem, including photosynthetic physiological characteristics (fluorescence parameters, pigment contents), gene expression and proteomic change. We want to examine the response of rice (Oryza sativaDN416), whose seeds were placed in Bio‐Radiation Box on the ShiJian‐10(SJ‐10) recoverable satellite. Our results demonstrated that the photosynthesis capacity of plants after spaceflight declined, compared to ground control plants. Specifically, Fv/Fm is significantly reduced for 7.5%. Chlorophyll content decreased in the three growth stages of rice, trefoil, tillering and mature stages. To further analyze changes under spaceflight environment, quantitative real‐time PCR technology and isobaric tags for relative and absolute quantization (iTRAQ) labeling technology were deployed. We found that the gene expression of important subunits of key enzymes and important structures had been decreased after spaceflight. As for the results of changes in proteins, we discovered that the content of proteins related to electron transport and photosynthesis key enzyme declined. Our experiments can provide reference for further research to learn more about the effects of spaceflight on photosynthesis.