This book constitutes the refereed proceedings of the 21st International Conference on Implementation and Application of Automata, CIAA 2016, held in Seoul, South Korea, in July 2016. The 26 revised full papers presented were carefully reviewed and selected from 49 submissions. The papers cover a wide range of topics including characterizations of automata, computing distances between strings and languages, implementations of automata and experiments, enhanced regular expressions, and complexity analysis. .