Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of l-valine as a nutritional additive for all animal species. The production strain and its DNA were not detected in the final additive. Therefore, the final product does not give raise to any safety concern with regard to the genetic modification of the production strain. The EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) concludes that l-valine produced using Escherichia coli CCTCC M2020321 is safe when supplemented in appropriate amounts to the diet according to the nutritional needs of the target species. The FEEDAP Panel has concerns on the use of amino acids in water for drinking for hygienic reasons, and due to the risk of imbalances when administered simultaneously via feed. The use of l-valine produced using E. coli CCTCC M2020321 in animal nutrition is considered safe for the consumers and for the environment. The FEEDAP Panel cannot conclude on the potential of l-valine produced using E. coli CCTCC M2020321 to be toxic by inhalation, irritant to the skin or eyes, or a dermal sensitiser due to the lack of data. The endotoxin activity of the additive does not represent a hazard for users handling the additive when exposed by inhalation. The additive l-valine produced by fermentation using E. coli CCTCC M2020321 is regarded as an efficacious source of the essential amino acid l-valine for non-ruminant nutrition. For the supplemental l-valine to be as efficacious in ruminants as in non-ruminant species, it requires protection against degradation in the rumen.