To restore fertility of the F1 between Solanum macrocarpon and eggplant, amphidiploids were produced through doubling of chromosomes by colchicine treatment. Shoot tips and axillary buds of F1 plants were kept for 2 and 4 days in liquid Murashige and Skoog (MS) media with 0.05% colchicine. As a result of colchicine treatment, two amphidiploids were identified by observing root tip cell chromosome number, stomatal guard cell size, and pollen characteristics. The amphidiploids contained 48 chromosomes, twice the normal diploid number of 24. Stomata size and pollen diameter were significantly larger in amphidiploids than F1. Flower diameter and length and width of anther, petal, and sepal were significantly larger in the amphidiploids than in F1. Pollen stainability was 40% in amphidiploids but only 0.86% in the diploid F1. The amphidiploids, after selfing and backcrossing with S. macrocarpon, set fruits with healthy seeds. Therefore, production of amphidiploids by colchicine treatment restored the pollen and seed fertility of F1 between S. macrocarpon and eggplant. The amphidiploids produced in this study would be very useful in future breeding programs of eggplant.