<p>Histone variants are critical components of neuronal chromatin that are emerging as key regulators of long-term memory formation. We previously showed that depleting the macrodomain-containing histone variant macroH2A1 (mH2A1) in the mouse hippocampus impairs long-term memory, establishing this histone as essential for memory consolidation. However, mH2A1 undergoes alternative splicing to generate two isoforms, mH2A1.1 and mH2A1.2, which differ by a single exon within the macrodomain. Though mH2A1.1 and mH2A1.2 have been reported to regulate unique molecular processes in non-neuronal cells, distinct functional contributions of hippocampal mH2A1.1 and mH2A1.2 to long-term memory formation in the adult brain are unknown. Here, we characterized genomic localization of mH2A1 splice isoforms in the mouse hippocampus and evaluated how isoform-specific knockdown impacts hippocampal transcription and memory. Although both isoforms localize to and regulate memory-relevant genes, their depletion affected largely non-overlapping gene sets, and only loss of mH2A1.1 impaired long-term memory. Notably, mH2A1.1 depletion increased expression of several genes that negatively regulate memory formation, including the well-established memory suppressor calcineurin. Thus, under normal conditions, mH2A1.1 may promote memory by repressing transcriptional programs that constrain plasticity. Together, these findings reveal isoform-specific functions of mH2A1 in the hippocampus and identify alternative splicing of mH2A1 as a key epigenetic mechanism that fine-tunes neural chromatin composition to enable long-term memory formation.</p>