The impact of betaine on the development of hypertension remains unclear, and prospective data are sparse. We aimed to investigate the association of serum betaine with repeated measurements of blood pressure (BP) and hypertension incidence. This study was based on the Guangzhou Nutrition and Health Study (GNHS), a community-based prospective cohort study in China. Baseline serum betaine was measured by high-performance liquid chromatography-tandem mass spectrometry. BP and hypertension status were assessed at the baseline and 3-year intervals. Linear mixed-effects models (LMEMs) were used to analyze the longitudinal association of serum betaine with BP (n = 1996). Cox proportional hazard models were used to evaluate the association of baseline serum betaine with hypertension incidence (n = 1339). LMEMs showed that compared with the lowest quartile group, the higher quartile groups had lower systolic blood pressure (SBP), diastolic blood pressure (DBP) and pulse pressure (all P-trend < 0.05). Each standard deviation (16.3 μmol L-1) increase in serum betaine was associated with -0.92 (-1.52, -0.32) mmHg of SBP, -0.49 (-0.84, -0.13) mmHg of DBP and -0.43 (-0.81, -0.05) mmHg of pulse pressure. During a median follow-up of 9.2 years, 371 incident cases of hypertension were identified. Serum betaine was associated with lower risk of hypertension only when comparing the third quartile level with the lowest quartile (HR, 0.74; 95% CI, 0.56-0.99). A nonlinear association between serum betaine and the risk of hypertension was found (P-nonlinear = 0.040). A higher serum betaine level was associated with lower risk of hypertension below 54.5 μmol L-1. Our findings suggested that higher serum betaine was associated with favorable blood pressure in middle-aged and older Chinese adults. Higher concentrations of serum betaine were related to lower hypertension risk in people with relatively low serum betaine concentrations.