Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on l‐tryptophan produced by fermentation with a non‐genetically modified strain of Escherichia coli KCCM 10534 when used as a nutritional additive in feed and water for drinking for all animal species and categories. The production strain E. coli KCCM 10534 is safe for the production of l‐tryptophan and it was not detected in the final product. The use of l‐tryptophan produced using E. coli KCCM 10534 in supplementing feed to compensate for tryptophan deficiency in feedingstuffs is safe for non‐ruminant target species. There may be a risk for an increased production of toxic metabolites when unprotected tryptophan is used in ruminants. The FEEDAP Panel has concerns on the safety of the simultaneous oral administration of l‐tryptophan via water for drinking and feed due to possible amino acid imbalances. The use of l‐Tryptophan produced by E. coli KCCM 10534 in animal nutrition raises no safety concerns to consumers of animal products and to the environment. The additive under assessment is considered not toxic by inhalation, it is not a skin or eye irritant and is not a skin sensitiser. The endotoxin activity of the additive and its dusting potential indicate a risk by inhalation for the users. The product l‐tryptophan is regarded as an effective source of the amino acid l‐tryptophan for all non‐ruminant species. In order to be as efficacious in ruminants as in non‐ruminants, it should be protected from ruminal degradation.