Purpose: To study the resistance to third-generation cephalosporins in Providencia stuartii strain isolated from hospitalized patient in Tunisia and to identify the responsible genes Materials and Methods: This strain was analysed by PCR and sequencing to identify the genes responsible for the β-lactamase resistance phenotypes. The transferability of the phenotypes was tested by conjugation to Escherichia coli J53. The isoelectric point was determinate by isoelectrofocalisation. Results: This resistance was carried by a 60 kb plasmid that encoded a β-lactamase with a pI of 5.4. This β-lactamase revealed identity with the blaTEM-1 gene encoding the TEM-1 β-lactamase, except for a replacement of the Val residue at position 84 by Ile, and the Ala residue at position 184 by Val. These two mutations were encountered in TEM-116 β-lactamase. Conclusion: This study demonstrates the first description of TEM-116 in the P. stuartii species in the world and the first one in a Tunisian hospital.