Epidermolysis bullosa simplex (EBS) is a rare genodermatosis characterized by mechanical stress-induced blistering of the skin. Its pathogenesis is mainly associated with defects of attachment of basal keratinocytes to the underlying dermis, resulted from keratin (KRT) 5 and KRT 14 mutation. We experienced a sporadic case of EBS with a novel KRT 14 mutation. A 1-day-old female full-term newborn presented with multiple bullae and erosions mainly on hands and feet. She did not show signs of other systemic diseases. Familial history of dermatologic diseases was absent. The histopathologic examination showed intraepidermal cleft. Direct immunofluorescence finding was negative. Electromicroscopy showed that the level of cleavage was low within the basal keratinocytes, just above the level of the hemidesmosomes and keratin clumps were not observed. Additional genetic analysis by exon sequencing revealed a novel de novo heterozygous missense mutation c.369T>G (p.Asn123Lys) in KRT14 gene. On the other hand, other family members including parents and her brother did not show any abnormalities in KRT14 gene. From these findings, she was diagnosed with EBS. She is receiving conservative treatment and is being followed-up for 6 months without serious complications. Herein, we report a rare case of neonatal EBS with de novo KRT14 gene mutation, which has been only once reported in the literatures to our knowledge.