Conflicting evidence exists regarding the association of gut microbiota-related metabolites - TMAO and its precursor choline and betaine with type 2 diabetes Mellitus (T2DM), particularly in rural Chinese populations. This study aimed to prospectively examine these relationships in a northern rural Chinese cohort.A nested case-control study was conducted within the Handan Eye Study. At baseline (2006-2007), 5,512 participants aged ≥30 years were enrolled. After 6.5 years of follow-up, 209 incident T2DM cases and 394 age- and sex-matched controls were included. Plasma choline, betaine, and TMAO levels were measured using ultra-performance liquid chromatography. Logistic regression and linear models assessed associations with T2DM risk, insulin resistance (IR), and metabolic parameters.Baseline betaine level was lower in the T2DM group compared to controls (betaine: 7431.4 ng/mL versus 7821.5 ng/mL). After adjusting for BMI, waist-to-hip ratio, and diabetes history, no significant associations were found between choline, betaine, or TMAO and T2DM risk. However, higher betaine quartiles showed a trend toward reduced T2DM risk (Q4 vs Q1 OR: 0.59, 95% CI: 0.34-1.06). Betaine was inversely correlated with HOMA-IR (β = -0.16), HOMA-β (β = -0.13), and TyG index (β = -0.21) (p < 0.05). Plasma choline levels were positively associated with fruit intake frequency, while TMAO levels decreased with higher exercise intensity.Betaine may play a protective role against dyslipidemia, adiposity, and T2DM risk in rural Chinese populations. Further studies are needed to explore TMAO's complex role in diabetes development.© 2025 Liang et al.