In this study, we investigated the cellular functions of nuclear localizing protein (NLP) in Nicotiana benthamiana. The GFP fusion protein of Arabidopsis NLP is mostly targeted to the nucleus and cytosol, but it infrequently targets only the nucleus. Silencing of NLP using virus-induced gene silencing caused growth retardation and abnormal leaf development in N. benthamiana. At the cellular level, downregulation of NLP expression increased the size of pavement cells and stomata in the leaf tissues. NLP suppression also increased endoreduplication in leaf cells, suggesting a role of NLP in the endocycle. NLP transcript levels in leaves were significantly increased in response to infection with the nonhost pathogens. NLP deficiency attenuated HR cell death against nonhost pathogens and in response to gene-for-gene interaction. These findings suggest that NLP plays a role in plant development and plant defense responses against pathogens.