Insulin signaling pathway is conserved and important during the control of biological processes in organisms. In this study, using the simulated microgravity system in Caenorhabditis elegans, we examined the response of insulin signaling pathway to simulated microgravity. After simulated microgravity treatment for 24-h, we observed the significant decrease in expressions of daf-2 encoding an insulin receptor and two genes (age-1 and akt-1) encoding kinase proteins, and increase in expression of daf-16 encoding a FOXO transcriptional factor. Simulated microgravity also induced an obvious translocation and accumulation of DAF-16::GFP signals in the nucleus. Mutation of daf-2, age-1, or akt-1 caused the resistance to toxicity of simulated microgravity, and daf-16 mutation caused the susceptibility to toxicity of simulated microgravity, indicating the protective response of insulin signaling to simulated microgravity. RNA interference (RNAi) knockdown of daf-16 could suppress the resistance of daf-2, age-1, or akt-1 mutant nematodes to toxicity of simulated microgravity, implying the involvement of signaling cascade of DAF-2-AGE-1-AKT-1-DAF-16 in regulating the response to simulated microgravity. Tissue-specific activity analysis demonstrated that the insulin signaling pathway only acted in the intestine to regulate the response to simulated microgravity. Moreover, in the intestine, insulin signaling functioned synergistically with p38 mitogen-activated protein kinase (MAPK) signaling to regulate the response to simulated microgravity. Therefore, our results suggest the crucial role of intestinal insulin signaling pathway in response to simulate microgravity in nematodes. (C) 2019 Elsevier Inc. All rights reserved.