Iso-Migrastatin (iso-MGS) and lactimidomycin (LTM) are glutarimide-containing polyketide natural products (NPs) that are biosynthesized by homologous acyltransferase (AT)-less type I polyketide synthase (PKS) assembly lines. The biological activities of iso-MGS and LTM have inspired numerous efforts to generate analogues via genetic manipulation of their biosynthetic machinery in both native producers and model heterologous hosts. A detailed understanding of the MGS and LTM AT-less type I PKSs would serve to inspire future engineering efforts while advancing the fundamental knowledge of AT-less type I PKS enzymology. The mgs and ltm biosynthetic gene clusters (BGCs) encode for two discrete ATs of the architecture AT-enoylreductase (AT-ER) and AT-type II thioesterase (AT-TE). Herein, we report the functional characterization of the mgsB and ltmB and the mgsH and ltmH gene products, revealing that MgsB and LtmB function as type II thioesterases (TEs) and MgsH and LtmH are the dedicated trans-ATs for the MGS and LTM AT-less type I PKSs. In vivo and in vitro experiments demonstrated that MgsB was devoid of any AT activity, despite the presence of the conserved catalytic triad of canonical ATs. Cross-complementation experiments demonstrated that MgsH and LtmH are functionally interchangeable between the MGS and LTM AT-less type I PKSs. This work sets the stage for future mechanistic studies of AT-less type I PKSs and efforts to engineer the MGS and LTM AT-less type I PKS assembly lines for novel glutarimide-containing polyketides.