Controversy regarding the effects of betaine supplementation on cardiovascular markers has persisted for decades. This systematic review and meta-analysis compared the effects of betaine supplementation on cardiovascular disease (CVD) markers. Studies examining betaine supplementation on CVD markers published up to February 2021 were identified through PubMed, the Cochrane Library, Web of Science, Embase, and SCOPUS. Betaine supplementation had a significant effect on concentrations of betaine (MD: 82.14 μmol/L, 95% CI: 67.09 to 97.20), total cholesterol (TC) (MD: 14.12 mg/dl, 95% CI%: 9.23 to 19.02), low-density lipoprotein (LDL) (MD: 10.26 mg/dl, 95% CI: 6.14 to 14.38)], homocysteine (WMD: -1.30 micromol/L, 95% CI: -1.61 to -0.98), dimethylglycine (DMG) (MD: 21.33 micromol/L, 95% CI: 13.87 to 28.80), and methionine (MD: 2.06 micromol/L, 95% CI: 0.23 to 3.88). Moreover, our analysis indicated that betaine supplementation did not affect serum concentrations of triglyceride (TG), high-density lipoprotein (HDL), fasting blood glucose (FBG), C-reactive protein (CRP), liver enzymes [alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT)], and blood pressure. Our subgroup analysis suggested that a maximum dose of 4 g/d might have homocysteine-lowering effects without any adverse effect on lipid profiles reported with doses of ≥4 g/d. In conclusion, the present systematic review and meta-analysis supports the advantage of a lower dose of betaine supplementation (<4 g/d) on homocysteine concentrations without the lipid-augmenting effect observed with a higher dosage.