In plants, cysteine proteinase inhibitors (CPI or cystatin) are implicated in biotic and/or abiotic stress responses and developmental regulation. Using the techniques of RT-PCR and SMART RACE, full-length cDNAs of four CPI genes (NtCPI1, NtCPI2, NtCPI3, and NtCPI4) were cloned for the first time from Nicotiana tabacum L. cv. K326. The four sequences have been deposited in GenBank, with accession numbers KF057988, KF057989, KF057990, and KF057991, respectively. Genomic DNA sequence analysis showed that NtCPI1 and NtCPI2 each have a single intron, while the other two have no intron. The four genes encoded predicted proteins of 98, 98, 120, and 123 amino acid residues, respectively. In addition to the typical inhibitory motifs, namely the central signature motif QXVXG, a GG doublet in the N-terminal region, and A/PW residues in the C-terminal part, these deduced amino acid sequences contained the PhyCys-specific LARFAV-like motif in the N-terminal region, of which an N-terminal signal peptide of 27 residues was found in both NtCPI3 and NtCPI4. Messenger RNAs specific to the four genes were detected in roots, stems, leaves, and buds by semi-quantitative RT-PCR, which indicated that they were broadly expressed in tobacco. This study lays the foundation for further exploration of the physiological functions of these cysteine proteinase inhibitor genes in plants.