Objective: To investigate the impact of metabolic labeling on Porphyromonas gingivalis (Pg) and compare the imaging effects of two fluorescent probes. Methods: This study was reviewed by the unit Ethics Committee and was approved by the Experimental Animal Welfare Ethics Branch of the Unit Experimental Biomedical Ethics Committee. Pg integrated N-azidoacetylgalactosamine (Ac4GalNAz) via a bioorthogonal reaction and was labeled with Cy5-DBCO or Cy7-DBCO via a click chemistry reaction. The bacteria were divided into Pg group (control, not fluorescently labeled), Cy5-Pg group (tagged by Cy5-DBCO), and Cy7-Pg group (tagged by Cy7-DBCO). A live/dead staining kit was applied to test the viability of Pg, Cy5-Pg, and Cy7-Pg. The mRNA levels of interleukin-6 (IL-6) and IL-8 and cell proliferation were examined in human gingival fibroblasts (HGFs) after the challenge of Cy5-Pg, Cy7-Pg, or Pg. To investigate the stability of metabolic labeling, Cy5-Pg or Cy7-Pg was cocultured with Escherichia coli (E. coli). Cy5-Pg and Cy7-Pg signal intensity with serial dilutions were examined using an in vivo imaging system (IVIS). Finally, C57BL/6J mice were orally administered Cy5-Pg or Cy7-Pg for IVIS detection, and the signal-to-background ratios were calculated. Results : Metabolic labeling could be applied to label live Pg in vitro. The optimal labeling concentrations for Cy5 and Cy7 were 20 μmol/L and 30 μmol/L, respectively. The area ratios of live to dead bacteria were approximately 2.0 in the three groups (F = 0.318, P>0.05). After a 6-h challenge with Cy5-Pg, Cy7-Pg, or Pg, the mRNA levels of HGFs increased by 7.86-, 7.46-, and 6.56-fold for IL-6, respectively (F = 40.886, P<0.001) and 12.43-, 13.03-, and 13.71-fold for IL-8 (F = 18.781, P<0.01), were spectively, compared to that of the Ctrl group, which was not challenged by bacteria, where no significant differences were observed among the three groups (P>0.05). HGFs were further challenged by Cy5-Pg, Cy7-Pg, or Pg at different MOIs, and cell proliferation was significantly inhibited (MOI = 104∶1, F = 153.52, P<0.001; MOI = 105∶1, F = 331.21, P<0.001; MOI = 106∶1, F = 533.65, P<0.001), with no significant differences among the three groups (P>0.05). Within 24 h of co-culturing Cy5-Pg or Cy7-Pg with E. coli, minimal E. coli was detected. The intensities of Cy5 and Cy7 remained stable for 3 h. Additionally, the fluorescence signal intensities of Cy5 and Cy7 were linearly correlated with the concentration (R2 = 0.97). After oral gavage of Cy5-Pg or Cy7-Pg in mice for the abdominal region at 1 h and 3 h, the signal-to-background ratios of Cy7-Pg were approximately 4.24-fold (t = 6.893, P<0.01) and 3.77-fold (t = 4.407, P<0.05) higher, respectively, than those of Cy5-Pg. For the isolated gastrointestinal tracts at 3 h, the signal-to-background ratio of Cy7-Pg was 5.19-fold higher than that of Cy5-Pg (t = 4.418, P<0.05). Conclusions Metabolic labeling did not significantly affect viability, immunomodulatory ability, and toxicity. The imaging effect of Cy7 on IVIS was better than that of Cy5. Our study provided experimental evidence for the correlation between periodontitis and overall health.