Objective ·To employ a two-sample Mendelian randomization (MR) design to examine whether the associations between loneliness, subjective well-being, and the frailty index reflect causal relationships. Methods ·Summary data from published genome-wide association studies (GWAS) were utilized, and an MR approach was employed to analyze the causal relationships between loneliness, subjective well-being, and the frailty index (as a surrogate measure of frailty). Data on loneliness were derived from a study comprising 445 024 individuals of European ancestry, including 80 134 cases and 364 890 controls. Data for subjective well-being were obtained from 298 420 participants of European ancestry. Summary statistics for frailty were sourced from a meta-analysis of GWAS conducted within the UK Biobank and the Swedish TwinGene cohort, which included 175 226 individuals of European ancestry. The inverse variance weighted (IVW) method served as the primary MR analytical approach to derive causal estimates, with the weighted median estimator (WME) used as a supplementary approach. Cochran′s Q test was applied to assess heterogeneity in the IVW estimates. The presence of horizontal pleiotropy was evaluated using the MR-Egger intercept test. When significant heterogeneity was detected, the MR-PRESSO global test was utilized to identify and subsequently remove outlier variants. MR analyses were then repeated. A Leave-One-Out sensitivity analysis was conducted to evaluate whether the overall estimates were unduly influenced by any single nucleotide polymorphism (SNP). To ensure data consistency and analytical reliability during effect allele harmonization between exposure and outcome datasets, palindromic SNPs (for which the effect allele direction could not be definitively determined) were excluded. Results ·The IVW method revealed a significant positive association between loneliness and the frailty index, with an OR of 3.87 (95% CI 2.33‒6.46, P<0.001). This result was further confirmed by the WME, yielding an OR of 2.81 (95% CI 1.49‒5.29, P<0.001). Subjective well-being showed a significant negative association with the frailty index (OR=0.80, 95% CI 0.69‒0.94, P=0.005). The WME demonstrated a consistent direction of effect, though with borderline significance (OR=0.86, 95% CI 0.74‒1.01, P=0.065). The analysis revealed no significant heterogeneity (Cochran′s Q test, P=0.054 and P=0.074) or horizontal pleiotropy (MR-Egger intercept test, P=0.470 and P=0.260). Conclusion ·This study, supported by the MR methodology, indicates that loneliness is associated with an increased risk of frailty, while subjective well-being serves as a protective factor against frailty.