Aims: To purify and characterize an exo-acting chitinolytic enzyme produced from a Gram-negative bacterium Pseudomonas fluorescens JK-0412. Methods and Results: A chitinolytic bacterial strain that showed confluent growth on a minimal medium containing powder chitin as the sole carbon source was isolated and identified based on a 16S ribosomal DNA sequence analysis and named Ps. fluorescens JK-0412. From the culture filtrates of this strain, a chito-oligosaccharides-degrading enzyme was purified to apparent homogeneity with a molecular mass of 50 kDa on SDS-PAGE gels. The kinetics, optimum pH and temperature, and substrate specificity of the purified enzyme (named as NagA) were determined. Conclusions: An extracellular chitinolytic enzyme was purified from the Ps. fluorescens JK-0412 and shown to be an exo-type beta-N-acetylglucosaminidase yielding GlcNAc as the final product from the natural chito-oligosaccharides, (GlcNAc)(n), n = 2-5. Significance and Impact of the Study: As NagA is secreted extracellularly in the presence of colloidal chitin, Ps. fluorescens JK-0412 can be recognized as a potent producer for industry-level and cost-effective production of chitinolytic enzyme. This enzyme appears to have potential applications as an efficient tool for the degradation of chitinous materials and industry-level production of GlcNAc. To the best of our knowledge, this is the first report on an exo-type chitinolytic enzyme of Pseudomonas species.