The egg stage is an important stage in the silkworm (Bombyx mori) life cycle. Normal silkworm eggs are usually short, elliptical, and laterally flattened, with a sometimes hollowed surface on the lateral side. However, the eggs laid by homozygous recessive “Ming” lethal egg mutants (l-em) lose water and become concaved around 1h, ultimately exhibiting a triangular shape on the egg surfaces. We performed positional cloning, and narrowed down the region containing the gene responsible for the l-em mutant to 360kb on chromosome 10 using 2287 F2 individuals. Using expression analysis and RNA interference, the best l-em candidate gene was shown to be BmEP80. The results of the inverse polymerase chain reaction showed that an ~1.9kb region from the 3′ untranslated region of BmVMP23 to the forepart of BmEP80 was replaced by a >100kb DNA fragment in the l-em mutant. Several eggs laid by the normal moths injected with BmEP80 small interfering RNAs were evidently depressed and exhibited a triangular shape on the surface. The phenotype exhibited was consistent with the eggs laid by the l-em mutant. Moreover, two-dimensional gel electrophoresis showed that the BmEP80 protein was expressed in the ovary from the 9th day of the pupa stage to eclosion in the wild-type silkworm, but was absent in the l-em mutant. These results indicate that BmEP80 is responsible for the l-em mutation.