The precise control of cell cycle progression is critical for coherent development. In all eukaryotes, the cell cycle is controlled by complexes composed of a cyclin-dependent kinase (CDK) and a cyclin. CDK activity is controlled at multiple levels, including association with CDK inhibitory proteins called CKIs. Here, we report the isolation and characterisation of a novel Nicotiana tabacum CKI, named NtKIS2, revealing the existence of a CKI family in tobacco. Like NtKIS1a, the tobacco CKI we previously identified, the NtKIS2 protein interacts with A-type CDK and D-type cyclins; is localised in the nucleus; and its overexpression strongly impairs plant development. Furthermore, our results show that NtKIS2 is a cell division inhibitor in planta and suggest that this CKI acts mainly in G1 phase. However, NtKIS2 shows clear differences to NtKIS1a in its expression patterns both during the cell cycle and plant development. Finally, to understand the developmental modifications seen in planta, the links between cell division inhibition and stomata determination or chloroplast division are explored.