In order to develop a new male-sterile line of eggplant (Solanum melongena L.), the F1 rootstock ‘Assist’ (Solanum aethiopicum Aculeatum L. Group × S. melongena ‘DMP’) was continuously backcrossed to S. melongena ‘Uttara’ using ‘Uttara’ as a recurrent pollen parent up to BC4 generation. All examined F1 ‘Assist’ produced pollen grains while BC1 plants segregated into male fertile and sterile plants. The male-sterile plant did not produce any pollen grains. BC2, BC3, and BC4 progenies obtained from male fertile backcross progenies were segregated into male fertile and sterile plants and each segregation ratio fitted 3 : 1. BC3 and BC4 progenies obtained from male-sterile backcross progenies were fixed to male-sterility without segregation. Selfed progenies of the male fertile BC2 progeny were segregated into male fertile and sterile plants and the segregation ratio fitted 15 : 1. Therefore, the pollen non-formation characteristic is assumed to be a form of cytoplasmic male-sterility (CMS), induced by incompatibility between the cytoplasm of S. aethiopicum Aculeatum Group and the nucleus of S. melongena. Further, these segregation results indicated that two independent dominant fertility restorer (Rf) genes control the fertility restoration of this CMS. High values of fruit set, number of seeds per fruit, and the seed germination rate found in BC4 progenies indicate that the cytoplasm of S. aethiopicum Aculeatum Group has no notable negative effect on seed fertility of S. melongena. A new male-sterile line of eggplant could be developed by utilizing the cytoplasm of S. aethiopicum Aculeatum Group.