Fimbriae play an important role in establishing an infection by adhering to specific host tissues. The available genome sequences of many enterobacteria reveal multiple fimbrial loci are commonly contained in a genome. In addition to type 1 and 3 fimbrial gene clusters, seven novel fimbrial operons were identified in Klebsiella pneumoniae NTUH K2044 and named kpa, kpb, kpc, kpd, kpe, kpf and kpg. We have previously generated a type 3 fimbrial display system by replacing the mrkD adhesin gene with each of the fimbrial adhesion genes. In the study, analysis using immuno-fluorescence microscopy (IFM) and western blot hybridization were employed to demonstrate the recombinant type 3 fimbriae expressed properly. The recombinant fimbriae was found to be able to bind to erythrocytes of guinea pig, and the hemaaglutination (HA) activity was inhibited by 0.002 M D-mannose. In addition, the recombinant plasmid for overexpression of KpfD was generated and transformed into Escherichia coli NovaBlue (DE3). The heterologous expressed KpfD protein was then purified for polyclone antibody preparation. The antibody was then used to demonstrate the KpfD was assembled on the tip of the recombinant type 3 fimbriae by western blot and IFM analysis. The HA competition analysis revealed that the agglutination activity could be inhibited by 0.001 M D-mannose or 0.01 M N-acetyl-D-glucoseamind, but not affected by D-fucose. Furthermore, the KpfD antibody could inhibit the HA activity indicating KpfD adhesin is responsible for the specific binding activity to erythrocytes of guinea pig. Comparative analysis of the secondary and tertiary structure with E. coli FimH revealed a putative mannose binding pocket on KpfD D73 to A83.The recombinant plasmids containing truncation of the KpfD protein with or without the predicted mannose binding pocket were generated and the recombinant proteins purified for competition assay of the HA activity. However, neither recombinant protein could inhibit the HA activity. Cell adherence analysis indicated that the overexpression of the type 3 fimbriae on the surface of the recombinant E. coli rendered an autoaggregation phenotype. In order to prevent from the overestimated non-specific cell binding activity, the recombinant plasmid containing kpfABCD or kpfRABCD was generated and the expression of the recombinant kpf fimbriae was assessed by western blot hybridization, IFM assay, and HA activity measurement. The results indicated that the recombinant E.coli expressed kpf fimbriae on the surface exerted an HA activity that could be inhibited by D-mannose.