Current first-line medications for sleep disorders often overlap with antidepressants, yet their effectiveness remains suboptimal, highlighting the urgent need for novel therapeutic strategies based on new mechanisms. Our study initially identified a significant reduction in serum S-adenosylmethionine (SAM) levels in insomnia patients through a cross-sectional analysis, suggesting SAM as a potential biomarker and therapeutic target for sleep disorders. We then screened 60 gut strains across seven species and identified a high-SAM-producing probiotic, Lactobacillus helveticus CCFM1320, which improved cognitive and memory impairments caused by sleep deprivation in mice. Mechanistically, CCFM1320 enhances the methylation of N-acetylserotonin, a precursor of melatonin synthesis, normalizing the expression of downstream circadian rhythm genes. A subsequent four-week placebo-controlled clinical trial demonstrated that CCFM1320 significantly improved sleep quality in patients with sleep disorders, as evidenced by reduced Pittsburgh sleep quality index (PSQI) scores, lower serum cortisol levels, and decreased prevalence of pathogenic species in the gut. Probiotic treatment also elevated the abundance of SAM synthesis and metabolism-related enzyme genes in the gut microbiome, likely due to the introduction of high-SAM-producing probiotics and subsequent SAM accumulation. This study presents a promising probiotic-based strategy for managing sleep disorders, offering a potential non-pharmacological treatment alternative.