Glycine betaine (betaine) acts as a methyl group donor in transmethylation reactions in organisms. Betaine occurs in numerous vertebrate tissues as an osmolyte, ensuring osmoprotection. Betaine is safe for piglets at the maximum supplementation rate of 2 000 mg/kg complete feed with a margin of safety below 5. This conclusion is extended to all pigs and extrapolated to all animal species and categories. The use of betaine as a feed additive up to a supplementation rate of 2 000 mg/kg complete feed is unlikely to pose concerns for consumer safety. Users’ inhalation exposure to betaine is expected to be minimal. Betaine anhydrous should be considered irritant to skin, eyes and mucous membranes and a skin sensitiser. It is likely to cause skin sensitisation. The supplementation of feed with betaine anhydrous does not pose a risk to the environment. Betaine has the potential to become efficacious in all animal species and categories when administered via feed or water for drinking. The FEEDAP Panel made some recommendations on (i) introduction of a maximum content for supplemental betaine in complete feed and water for drinking; (ii) avoidance of simultaneous use of betaine in feed and water for drinking; and (iii) avoidance of simultaneous inclusion of betaine and choline chloride in premixtures.