Lactate dehydrogenase is an enzyme of glycolytic pathway which catalyzes the interconversion of pyruvate and lactate. The present study describes cDNA cloning, E. coli expression and characterization of lactate dehydrogenase B (LDH-B) from the heart ventricles of river buffalo (Bubalus bubalis). Total RNA was isolated from the heart tissue, a 1005bp cDNA encoding complete polypeptide chain of 334 amino acids was generated by reverse transcriptase reaction and analyzed for nucleotide sequence. The consensus sequence obtained from both strands has shown 84% to 98% homology with that of different mammalian species. The attributed gene was cloned, expressed in BL21 (DE3) RIPL Codon Plus strain of E. coli using pET21a (+) plasmid. The purified recombinant enzyme displayed a KM value of 50 µM for pyruvate, an optimum activity at 35°C and pH 7.0. The enzyme was found as a homotetramer of 140 kDa on FPLC based gel-filtration column. Molecular weight of a subunit of enzyme as determined by mass spectrometric analysis was 36530.21 Da. The present study describes the first ever report about the cDNA sequence and characteristics of recombinant LDH-B from River buffalo.