Background: The association between physical activity (PA) and neuroticism has been established, but the genetic mechanism remains elusive now. Methods: In this study, 362,421 participants from UK Biobank were used to explore the associations between PA and neuroticism, using linear regression analysis. PA was measured as excess metabolic equivalent (MET)-hours per week, assessed from a combination of walking and moderate and vigorous PA. The Gene-environment-wide interaction analyses (GEWIS) was conducted by PLINK2.0 to evaluate gene×PA interacting effects on the risk of neuroticism. DAVID tool was then used to conduct gene ontology (GO) analysis of the observed PA-interacted genes. Results: We observed negative association between PA and neuroticism in total sample (b=−0.02, P=9.02×10−37), female sample (b=−0.02, P=5.74×10−28) and male sample (b=−0.02, P=6.17×10−14). GEWIS revealed multiple significant gene×PA interaction signals for neuroticism. 78 candidate genes were observed, such as ST18(rs76573348, P=6.45×10−16),NRCAM (rs117655943, P=4.28×10-11) and RBFOX3(rs113329299, P=4.72×10-10) in total group, SYT1(rs151016298, P=1.56×10−11), S100B(rs80199493, P=8.56×10−11) and MYT1(rs111360063, P=1.53×10−10) in female group, and NOTCH3 (rs184928292, P=3.31×10-17), SLC24A2 (rs62563282, P=9.56×10-16), CSMD1(rs73177368, P=2.11×10-13) in male group. GO enrichment analysis identified 15, 11 and 38 GO terms for total, female and male samples, such as synapse assembly (Ptotal=1.37×10-2), calcium ion binding (Pfemale=3.33×10-2) and learning or memory (Pmale=1.14×10-2).Conclusion: This study suggested the influences of physical activity on neuroticism, and identified several neuroticism associated genes interacting with PA.