Begomoviruses (family Geminiviridae) are widely distributed plant viruses and number of infected plants by this group of viruses is rapidly increasing due to adaptation of its vector in different geographical conditions and ability to recombine with other Geminiviruses. Begomovirus associated betasatellite DNA has a complementary sense transcript βC1, which is responsible for symptom induction. Antisense construct targeting βC1 gene was developed in suitable vector and transgenic Nicotiana tabacum plants were raised using this construct. No visible alteration was observed in the raised transgenic plants. Upon inoculation with viruliferous whiteflies (Bemisia tabaci) the raised βC1-anti transgenic plants of Nicotiana tabacum showed resistance against cotton leaf curl disease and no visible symptom was observed. Furthermore, the key component (small RNAs) was isolated from the transformed plants and hybridized with suitable probe. This study highlighted Antisense technology as an effective tool to develop resistance against cotton leaf curl disease (CLCuD).