Polyglutamine tract-binding protein-1 (PQBP1) is involved in the transcription-splicing coupling, and its mutations cause a group of human mental retardation syndromes. We generated a fly model in which the Drosophila homolog of PQBP1 (dPQBP1) is repressed by insertion of piggyBac. In classical odor conditioning, learning acquisition was significantly impaired in homozygous piggyBac-inserted flies, whereas the following memory retention was completely normal. Mushroom bodies (MBs) and antennal lobes were morphologically normal in dPQBP1-mutant flies. Projection neurons (PNs) were not reduced in number and their fiber connections were not changed, whereas gene expressions including NMDA receptor subunit 1 (NR1) were decreased in PNs. Targeted double-stranded RNA-mediated silencing of dPQBP1 in PNs, but not in MBs, similarly disrupted learning acquisition. NR1 overexpression in PNs rescued the learning disturbance of dPQBP1 mutants. HDAC (histone deacetylase) inhibitors, SAHA (suberoylanilide hydroxamic acid) and PBA (phenylbutyrate), that upregulated NR1 partially rescued the learning disturbance. Collectively, these findings identify dPQBP1 as a novel gene regulating learning acquisition at PNs.