Tomato leaf curl virus (ToLCV) disease is a serious threat for tomato cultivation in the tropics and subtropics. Despite serious efforts no immune commercial varieties or F(1) hybrids are available till date. In this study, the interaction between Solanum lycopersicum and ToLCV was characterized on molecular and biochemical basis. RNA silencing mediated by short interfering RNA (siRNA) and reactive oxygen species (ROS) has been proposed as central components of plant adaptation to several stresses. A comparative RNA interference study between two contrasting tomato genotypes, LA1777 (tolerant) and 15SBSB (susceptible) infected with Tomato Leaf Curl New Delhi Virus (ToLCNDV) revealed relatively higher accumulation of siRNA in the leaves of tolerant genotype. In LA1777, ToLCNDV produced chlorotic as well as necrotic areas at the inoculation sites 5-10 days post-inoculation. Caspase-9- and caspase-3-like activities were significantly increased in response to ToLCNDV infection in LA1777 at inoculated region. Activities of antioxidant enzymes involved in the detoxification of ROS were examined in both systemic and localized area of infection, and their expression level was further validated through quantitative real-time PCR of the corresponding transcripts. Expression patterns of three genes encoding pathogenesis-related proteins showed higher accumulation in tolerant genotype. Tolerance against the ToLCNDV in LA1777 can be attributed to the higher siRNA accumulation, localized cell death, altered levels of antioxidant enzymes and activation of pathogenesis-related genes at different durations of virus infection. Based on these direct and indirect evidences, we have proposed a putative mechanism for ToLCNDV tolerance in the tolerant genotype.