<jats:title>ABSTRACT</jats:title><jats:sec><jats:label /><jats:p>5‐Aminolevulinic acid (5‐ALA) has recently attracted attention for its potential applications in the fields of medicine and agriculture. In this study, <jats:italic>Corynebacterium glutamicum</jats:italic> was firstly engineered for 5‐ALA production via the C4 pathway. <jats:italic>HemA</jats:italic> encoding 5‐aminolevulinic acid synthase from <jats:italic>Rhodobacter sphaeroides</jats:italic> was codon optimized and expressed in <jats:italic>C. glutamicum</jats:italic> ATCC13032, resulting in accumulation of 5‐ALA. Deletion of all known genes responsible for the formation of acetate and lactate further enhanced production of 5‐ALA. Overexpression of <jats:italic>ppc</jats:italic> gene encoding phoenolpyruvate carboxylase resulted in an accumulation of 5‐ALA up to 2.06 ± 0.05 g/L. Furthermore, deletion of high‐molecular‐weight penicillin‐binding proteins (HMW‐PBPs) genes <jats:italic>pbp1a</jats:italic>, <jats:italic>pbp1b</jats:italic>, and <jats:italic>pbp2b</jats:italic> led to an increase in 5‐ALA production of 13.53%, 29.47%, and 22.22%, respectively. Finally, 5‐ALA production was enhanced to 3.14 ± 0.02 g/L in shake flask by heterologously expressing <jats:italic>rhtA</jats:italic> encoding threonine/homoserine exporter, and 86.77% of supplemented glycine was channeled toward 5‐ALA production in shake flask. The engineered <jats:italic>C. glutamicum</jats:italic> ALA7 strain produced 7.53 g/L 5‐ALA in a 5 L bioreactor. This study demonstrated the potential utility of <jats:italic>C. glutamicum</jats:italic> as a platform for metabolic production of 5‐ALA. Change of cell permeability by metabolic engineering HMW‐PBPs may provide a new strategy for biochemicals production in <jats:italic>Corynebacterium glutamicum</jats:italic>. Biotechnol. Bioeng. 2016;113: 1284–1293. © 2015 Wiley Periodicals, Inc.</jats:sec>