Actinoplanes missouriensis grows by forming branched substrate mycelia and produces terminal sporangia. Each sporangium contains a few hundred spores, which swim as zoospores after being released from sporangia. Previously, we disrupted 22 putative acyltransferase genes and examined their involvement in morphological differentiation. Here, we described the characterization of one of them, a putative 1-acyl-2-hydroxy-sn-glycerol-3-phosphate acyltransferase (AGPAT) encoded by plsC (AMIS_11360). The plsC null (∆plsC) mutant exhibited a conditional growth defect in a nutrient-poor medium. No differences were observed between the wild-type and ∆plsC strains in sporangium formation, spore release, or zoospore motility. We confirmed the AGPAT activity of PlsC; the recombinant polyhistidine-tagged PlsC protein transferred the acyl group from palmitoyl-coenzyme A to 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphate, resulting in the production of 1,2-dipalmitoyl-sn-glycero-3-phosphate.