Citrate synthase (CS) is a key enzyme of the tricarboxylic acid cycle and is widely distributed among prokaryotes and eukaryotes. Here, we report for the first time, the cloning, expression, and characterization of a novel CS from Streptomyces diastaticus No. 7 strain M1033 (SdCS). Gel filtration chromatography and matrix-assisted laser-desorption ionization–time-of-flight mass spectrometry (MALDI-TOF-MS) analyses indicate that SdCS forms homodimers with a molecular mass of approximately 100.0 kDa. The predicted amino acid sequence of SdCS is highly similar to those of bacterial homodimeric type I CSs. The pH and temperature optima for SdCS activity were 8.0 and 35 °C, respectively. The half-life (t1/2) of SdCS was 10 Min at 50 °C and was increased to 210 Min in the presence of oxaloacetate. The kinetic parameters of SdCS (kcat = 262.8 and 230.7 s−1; Km = 58.4 and 11.2 μM for acetyl-CoA and oxaloacetate, respectively) were comparable to those of dimeric CSs isolated from Gram-positive bacteria and eukaryotes. Moreover, SdCS activity was inhibited by ATP and ADP and stimulated by AMP. These findings provide a foundation for further investigations on the three-dimensional structure and mechanism of catalysis of SdCS.