Hemophilia A, an X-linked recessive bleeding disorder, is caused by mutations of F8 gene. In about 2% hemophilia A patients, no exonic mutation of F8 gene was found. We aimed to identify deep intronic mutations of F8 gene. We reanalyzed the next-generation sequencing data of six hemophilia A patients with negative F8 variant in either coding region or splice site. Deep intronic F8 c.5999-27A>G variant (NM_000132.3) was found in two unrelated moderate hemophilia A patients from different region, and one patient's mother was mild hemophilia A patient. Splice site prediction algorithms showed no impact of this variant on F8 mRNA splicing of exon 19, including Human Splicing Finder 3.1, NNSPLICE 0.9, NetGene2, and Transcript-inferred Pathogenicity score. Exonic splicing enhancer was predicted by ESEfinder, and no difference was found between the wild type and mutant sequence. The branch point predicted by SVM-BPfinder suggested that F8 c.5999-27A>G variant may disrupt the branch point in intron 18 and affect the acceptor site splicing of F8 exon 19. Sanger sequencing of F8 cDNA from peripheral blood mononuclear cells confirmed that F8 c.5999-27A>G variant caused F8 exon 19 skipping in proband and his mother. Skewed X chromosome inactivation was found in another X chromosome of this mother, combined with F8 c.5999-27A>G variant in trans. In conclusion, our study suggests that deep intronic F8 c.5999-27A>G variant may be responsible for F8 exon 19 skipping and lead to moderate hemophilia A. Systematic reanalysis of next-generation sequencing data could promote the diagnostic yields.