In the present paper, we report the biochemical characterization of a chromosomal toxin-antitoxin (TA) system in Mycobacterium tuberculosis, consisting of the Rv1991c gene and its upstream open reading frame (ORF) termed Rv1991a. Rv1991c was characterized as a toxin with ribonuclease activity and Rv1991a as the antitoxin against Rv1991c. Rv1991a interacted with Rv1991c to form a complex. A promoter located immediately upstream of Rv1991a was identified. Both Rv1991a and the Rv1991a-Rv1991c complex were able to bind to the promoter region of the Rv1991a-Rv1991c operon, indicating that the expression of the Rv1991a-Rv1991c operon can be autoregulated.