This study was performed to evaluate the microbiological profile and the calprotectin expression in gingival crevicular fluid (GCF) in spontaneous and experimentally induced gingival inflammation. Thirty-seven periodontally healthy subjects were evaluated in real life conditions (N-O gingivitis) as well as after 21days of experimental gingivitis trial (E-I gingivitis). During the experimental gingivitis trial, in one maxillary quadrant (test quadrant), gingival inflammation was induced by oral hygiene abstention, while in the contralateral (control) quadrant, oral hygiene was routinely continued. The results of the study showed that (1) the microbiological profile of quadrants where gingival inflammation was experimentally induced (i.e., E-I test quadrants) differed significantly from that of either quadrants where gingival inflammation was controlled by proper plaque control (i.e., E-I control quadrants) or quadrants with N-O gingivitis, and (2) GCF calprotectin was significantly higher at E-I test quadrants compared to either E-I control quadrants or quadrants with N-O gingivitis. A positive intrasubject correlation was found between GCF concentration of calprotectin at sites presenting N-O and E-I gingivitis. N-O and E-I gingivitis showed a different microbiological profile of the subgingival environment. GCF calprotectin is a reliable marker of gingival inflammation, and its concentration in N-O gingivitis is correlated with its expression in E-I gingivitis. The modality of plaque accumulation seems to affect the subgingival microbiological profile associated with a gingivitis condition. Calprotectin levels in GCF may be regarded as a promising marker of the individual susceptibility to develop gingival inflammation in response to experimentally induced plaque accumulation.