Peptidyl-prolyl cis-trans isomerases (PPIases) are a class of chaperone proteins that catalyze isomerization between the cis and trans configuration of a peptide bond. Flavobacterium johnsoniae, a model organism for the study of bacterial gliding motility and the Type 9 Secretion System (T9SS), has six different proteins that are predicted to encode PPIases. Loss of one putative PPIase (GldI) results in a complete lack of gliding motility. However, loss of another putative PPIase encoded by fjoh_4997 has no impact on motility. In this study, genes fjoh_2367 and fjoh_2368, which are immediately downstream of gldI and have protein products homologous to GldI, were deleted. We found that the mutants exhibited a decrease in speed as compared with the wild type (1.62 ± 0.77 μm/s for mutant 5 and 1.60 ± 0.67 μm/s for mutant 6 vs 2.02 ± 0.62 μm/s for wild type). The slope of the mean square displacement of single cells was lower for mutants as compared with the wild type (1.08 for mutant 5, and 1.13 for mutant 6 vs 1.48 for wild type). Additionally, mutant colonies exhibited less spreading on PY2 agar plates than wild type, as shown by their measured colony diameters, which were 22.91 ± 2.18 mm for wild type, 16.75 ± 2.17 mm for mutant 5, and 15.97 ± 1.12 mm for mutant 6.