The production of 1,3-butanediol (1,3-BDO) from glucose was investigated using Escherichia coli as the host organism. A pathway was engineered by overexpressing genes phaA (acetyl-CoA acetyltransferase), phaB (acetoacetyl-CoA reductase), bld (CoA-acylating aldehyde dehydrogenase), and yqhD (alcohol dehydrogenase). The expression levels of these genes were optimized to improve 1,3-BDO production and pathways that compete with 1,3-BDO synthesis were disrupted. Culture conditions were also optimized, including the C: N ratio, aeration, induction time, temperature, and supplementation of amino acids, resulting in a strain that could produce 1,3-BDO at 257 mM in 36 h, with a yield of 0.51 mol/mol in a fed-batch bioreactor experiment. To the best of our knowledge, this is the highest titer of 1,3-BDO production ever reported using biological methods, and our findings provide a promising strategy for the development of microbial cell factories for the sustainable synthesis of other acetyl-CoA-derived chemicals.