Background: Circulating S-Klotho is increasingly recognized as an anti-aging protein linked to cardiovascular protection and metabolic regulation. Although exercise elevates S-Klotho, the dose–response threshold and metabolic determinants of this response in young adults with obesity, a population at heightened risk for cardiometabolic dysfunction, remain poorly characterized. To examine the acute effects of submaximal and maximal cycling on circulating S-Klotho in young adults with obesity and to identify key metabolic predictors of exercise-induced changes. Methods: Eighteen young (26 ± 3 years, BMI 30 ± 2 kg/m²) males and females completed incremental cycling to exhaustion. Serum S-Klotho was assessed at rest, after submaximal (SubMax), and post-maximal (Max) exercise. Covariates included cardiorespiratory fitness (VO_2max), iDXA body composition, HOMA-IR, and hsCRP. Predictor Screening and Partial Least Squares (PLS) regression with fivefold cross-validation were used to identify determinants of exercise-induced S-Klotho responses. Results: A significant acute increase in S-Klotho was observed (Main Effect of Time: F_(2,32) = 9.4, p = 0.0006). Post hoc analysis revealed an intensity-dependent threshold, with a significant surge occurring only between submaximal and maximal exertion (p = 0.021; Rest to Max: p = 0.0005). No significant sex differences or interactions were observed (p > 0.05). The PLS model explained 74% of the variance in S-Klotho changes, identifying systemic inflammation (Log₁₀[hsCRP]) and visceral adiposity as the primary predictive factors. Conclusions: Exhaustive exercise acutely increases circulating S-Klotho in young adults with obesity, independent of sex. This response is intensity-dependent and primarily driven by baseline metabolic risk factors. S-Klotho may represent an acute exercise-responsive biomarker linked to metabolic health in at-risk populations. These exploratory findings warrant confirmation in larger and adequately powered studies.