Background: Pirins are nuclear cupin proteins, one of several gene families within the plant cupin superfamily. However, the identification and functional analysis of Pirin proteins in Nicotiana benthamiana have not been explored. Methods: In this study, genome-wide analysis identifying NbPirin genes in N. benthamiana was conducted, as was phylogenetic analysis of Pirin genes in four Solanaceae species (including Capsicum annuum, Solanum lycopersicum, Solanum tuberosum, and N. benthamiana). In addition, we also evaluated the expression pattern of NbPirins under abiotic stress (temperature and phytohormones) and biotic stress (TMV, TuMV, and PVX). Results: A total of six Nbpirin genes were identified, which can be divided into three clades, and NbPirins also embraced a variety of abiotic or biotic cis-acting elements. The results showed that the expression of NbPirin1-6 was influenced by temperature variations, of which NbPirin6 was significantly upregulated at high temperatures (42 °C) but downregulated at low temperatures (4 °C). Notably, the expression of NbPirin6 exhibited a consistent decrease under ABA and MeJA treatments. Moreover, the expression of NbPirin1-6 was also affected by TMV, TuMV, and PVX infection. NbPirin1, NbPirin2, NbPirin3, and NbPirin5 showed higher expression levels under different viral infections compared to non-infection. Interestingly, NbPirin3 showed the highest expression level during TuMV infection (approximately a 20-fold increase compared to non-infection). Conclusions: Our study proposes the potential role of NbPirin6 in plant responses to abiotic stress, and the role of NbPirin3 in plant antiviral defense, and further lays the groundwork for future research on the functions of NbPirin proteins in responses to various stressors.