Purpose <p>Betaine is connected to various cardio-metabolic outcomes, yet its relationship with hyperuricemia remains uncertain. We aimed to longitudinally investigate the association between serum betaine levels and the risk of hyperuricemia in middle-aged and older adults.</p> Methods <p>This study utilized data from the Guangzhou Nutrition and Health Study (GNHS). Participants were enrolled between 2008 and 2010, with follow-ups conducted every three years, comprised an analysis sample of 2204 adults aged 40–75. Baseline serum betaine levels were assessed using high-performance liquid chromatography-tandem mass spectrometry. Serum uric acid (SUA) levels were measured at baseline and every three years thereafter. Linear mixed-effects models (LMEMs) and generalized estimating equations (GEEs) were employed to examine the longitudinal association between serum betaine levels and SUA levels, as well as hyperuricemia risk, respectively.</p> Results <p>LMEMs indicated that compared to individuals in the lowest quartile (Q1) of serum betaine levels, those in the highest quartile (Q4) exhibited the lowest SUA levels (Q4 vs. Q1: β -8.19, 95% CI -16.32 to -0.06, <i>P</i>-trend = 0.023). Each standard deviation increase in betaine (16.5 µmol/L) was associated to SUA levels decrease of -3.28 (-6.10, -0.45). Results from the GEE model suggested a 19% reduction in the odds of hyperuricemia (OR = 0.81; 95% CI: 0.68, 0.95, <i>P</i>-trend = 0.051) in the Q4 group compared to the Q1 category.</p> Conclusion <p>Our findings highlight a negative association between serum betaine levels and SUA levels, as well as the risk of hyperuricemia in middle-aged and older adults, which is more pronounced in individuals with better dietary quality.</p> Trial registration <p>This trial was registered at clinicaltrials.gov as NCT03179657. Website: <a href="https://clinicaltrials.gov/ct2/show/NCT03179657">https://clinicaltrials.gov/ct2/show/NCT03179657</a></p>