Birt-Hogg-Dubé syndrome (BHD) was an autosomal dominant disorder caused by a mutation in the folliculin () gene and characterized by benign cutaneous fibrofolliculomas in the head and neck, pulmonary cysts, spontaneous pneumothorax, and combined renal tumors. This study reported a familial case presenting multiple pulmonary bullae, recurrent spontaneous pneumothorax, diffuse cystic lesions in both lungs, and renal cysts. To further clarify the diagnosis, next-generation sequencing (NGS) was performed in conjunction with the clinical diagnostic criteria for Birt-Hogg-Dubé. The eukaryotic recombinant expression vectors of pEGFP-C1- and knock-in mutation by CRISPR/Cas9 were conducted in 293 T and BEAS-2B cell lines. The mRNA and protein expression of the mutation were verified by fluorescence quantitative PCR and Western blot assay. Nonsense-mediated mRNA decay (NMD) assays and immunohistochemical assays were conducted to elucidate the pathogenicity of the mutation and explore potential mechanisms. A unique, novel, unspecified significance mutation NM_144997.7: c.21_22del (p. Cys8 Profs⁣28) in Exon 4 was detected in both patients. The results demonstrated that the newly identified frameshift mutation significantly decreased mRNA and protein expression. The NMD complex recognized and degraded mRNAs containing a premature termination codon (PTC) in the open reading frame of the frameshift mutation, resulting in haploinsufficiency and ultimately contributing to the manifestation of BHD. Protein expression on the AMP-activated protein kinase (AMPK), Wnt/-catenin, and mammalian target of rapamycin (mTOR) signaling pathways by immunohistochemistry indicated that frameshift mutations were responsible for BHD through the activation of AMPK, Wnt/-catenin, and mTOR signaling pathways. The study demonstrated that a novel frameshift mutation was responsible for the pathogenesis of BHD and preliminarily demonstrated that causes BHD through the AMPK, Wnt/-catenin, and mTOR signaling pathways.