BACKGROUND: Premature ovarian insufficiency (POI) is a refractory reproductive disorderfeatured by accelerated follicular atresia and granulosa cells (GCs) apoptosis, yet the underlying metabolic-epigenetic mechanism remains unclear. This study aimed to explore the role of protein lactylation in follicular atresia during the progression of POI. METHODS: A cyclophosphamide-induced POI rat model was established and treated with nicotinamide mononucleotide (NMN) or estradiol (E2). Estrous cycle, ovarian morphology, reproductive hormone levels and follicle counts were evaluated. RNA-sequencing, qRT-PCR, Western blotting, TUNEL assay and immunofluorescence were used to detect cell apoptosis, glycolysis and related gene expression. Activity-based protein profiling (ABPP), co-immunoprecipitation, LC-MS/MS and site-directed mutagenesis were applied to identify lactate target proteins and lactylation sites. Cx43 hemichannel activity was measured by ethidium bromide uptake assay. Data were analyzed using unpaired t-test or one-way ANOVA followed by Bonferroni's post-hoc test. RESULTS: NMN treatment restored estrous cyclicity, decreased serum follicle-stimulating hormone (FSH) level, increased anti-Müllerian hormone (AMH) and estradiol (E2) levels, and inhibited granulosa cell apoptosis and oxidative stress in POI rats. NMN elevated ovarian NAD+ level, upregulated glycolytic rate-limiting enzymes (HK2, PKM2, LDHA) and restored lactate production. Lactate enhanced Cx43 expression and hemichannel activity. GRP78 was identified as a direct target protein of lactate, which was lactylated at lysine 352(K352) by the acetyltransferase P300. GRP78 lactylation maintained Cx43 expression and lactate transport function, while the K352R mutation impaired Cx43 activity, aggravated granulosa cell apoptosis and oxidative stress. CONCLUSION: NMN promotes glycolysis and lactate production by replenishing NAD+. Lactate-induced GRP78 lactylation at K352 maintains Cx43 function and suppresses follicular atresia. Lactylation of GRP78 provides a novel molecular mechanism and potential therapeutic target for POI.