Depolymerase is an enzyme that plays an important role in the hydrolysis of polyhydroxyalkanoates [PHAs]. In the current study, Burkholderia cepacia DP1 was obtained from Penang, Malaysia in which the enzyme was purified using ion exchange and gel filtration (Superdex-75) column chromatography. The molecular mass of the enzyme was estimated to be 53.3 kDa using SDS-PAGE. The enzyme activity was increased to 36.8 folds with the recovery of 16.3% after purification. The enzyme activity was detected between pH 6.0-10 and at 35-55 degrees C with pH 6.0 and 45 degrees C facilitating the maximum activity. Depolymerase was inactivated by Tween-20, Tween-80, SDS and PMSF, but insensitive to metal ions (Mg2+, Ca2+, K+, Na2+, Fe3+) and organic solvents (methanol, ethanol, and acetone). The apparent K-m values of the purified P(3HB) depolymerase enzyme for P(3HB) and P(3HB-co-14%3HV) were 0.7 mg/ml and 0.8 mg/ml, respectively. The V-max values of the purified enzyme were 10 mg/min and 8.89 mg/min for P(3HB) and P(3HB-co-14%3HV), respectively. The current study discovered a new extracellular poly(3-hydroxybutyrate) [P(3HB)] depolymerase enzyme from Burkholderia cepacia DP1 isolated and purified to homogeneity from the culture supernatant. To the best of our knowledge, this is the first report demonstrating the purification and biochemical characterization of P(3HB) depolymerase enzyme from genus Burkholderia.