Aim To investigate the effect of lncRNA NEAT1 on myocardial fibrosis in rats with atrial fibrillation (AF) by regulating miR-27b-3p/specific protein 1 (SP1) axis. Methods 54 rats were divided into six groups (n＝9) according to the random number table method:sham group, AF group, AF＋shControl group, AF＋shNEAT1 group, AF＋shNEAT1＋anti-miR-Control group and AF＋shNEAT1＋anti-miR-27b-3p group. After 2 weeks of transfection with the above lentiviral vector, AF rats was established by tail vein injection of acetylcholine-CaCl₂ for 7 days. The incidence and duration of AF were recorded by electrocardiogram; the expression levels of NEAT1, miR-27b-3p, SP1 and fibrosis-related genes (TGF-β1, CTGF, COLⅠ, COLⅢ) in atrial tissue were detected by RT-qPCR; the luciferase report gene confirmed the targeting relationship between miR-27b-3p and NEAT1, and miR-27b-3p and SP1; cardiomyocyte apoptosis in atrial tissue was observed by TUNEL staining; myocardial fibrosis in atrial tissue was observed by Masson staining; and the protein expression levels of SP1 and fibrosis-related genes in atrial tissue were detected by Western blot. Results Compared with the AF group, the incidence and duration of AF, myocardial fibrosis, apoptosis and the mRNA and protein expression of TGF-β1, CTGF, COLⅠ and COLⅢ were significantly decreased in AF＋shNEAT1 group (P＜0.05). NEAT1 negatively regulates the expression of miR-27b-3p. Silencing miR-27b-3p reversed the inhibitory effects of shNEAT1 on myocardial apoptosis and fibrosis in AF rats. miR-27b-3p directly targeted SP1 and inhibited its mRNA and protein expression. NEAT1 promoted SP1 expression by down-regulating miR-27b-3p. Conclusion Down-regulation of NEAT1 can alleviate AF and AF-induced myocardial fibrosis, and its regulatory mechanism is related to the regulation of miR-27b-3p/SP1 axis.