In 2017, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) concluded that betaine as a novel food (NF) is safe to be used at a maximum intake level of 6 mg/kg body weight per day in addition to the intake from the background diet. Following the present request from the European Commission, the EFSA NDA Panel was asked to deliver a scientific opinion on betaine by carrying out the assessment for the revised uses and use levels of betaine as proposed by the applicant. Thus, EFSA performed an intake assessment based on individual data from the EFSA Comprehensive European Food Consumption Database and the new proposed uses of the NF for the general population. The resulting ranges for the mean and high-level estimated intakes of betaine for the general population do not exceed the safe level of intake previously established. The Panel concludes that the NF, betaine, is safe under the new proposed conditions of use.