The start-up of microbial electrohydrogenesis cells (MECs) is a key-step to realize efficient biohydrogen generation and adequate, long-term operation. This review paper deals with the lessons and experiences reported on the most important aspects of H 2 producing MEC start-up. The comprehensive survey covers the assessment and discussion of the main influencing factors and methods (e.g. inocula selection, enrichment, acclimation, operating conditions and cell architecture) that assist the design of MECs. This work intends to be a helpful guide for the interested readers about the strategies employed to successfully establish microbial electrochemical cells for sustainable biohydrogen production.