Periodontitis is the chronic inflammatory destruction of the periodontal tissues, as a result of bacterial biofilm formation on the tooth surface. Proteins secreted by the gingival epithelium challenged by subgingival biofilms represent an important initial response for periodontal inflammation. The aim of this in vitro study was to characterize the whole secreted proteome of gingival epithelial tissue challenged by subgingival biofilms, and to evaluate the differential effects of the presence of the red complex species in the biofilm. Multi-layered human gingival epithelial cultures were challenged with a 10-species in vitro biofilm model or its 7-species variant excluding the red complex. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) for label-free quantitative proteomics was applied to identify and quantify the secreted epithelial proteins in the culture supernatant. A total of 192 proteins were identified and quantified. The biofilm challenge resulted in more secreted proteins being down-regulated than up-regulated. Even so, presence of the red complex in the biofilm was responsible for much of this down-regulatory effect. Over 24 h, the up-regulated biological processes were associated with inflammation and apoptosis, whereas the down-regulated ones with the disruption of epithelial tissue integrity and impairment of tissue turnover. Over 48 h, negative regulation of several metabolic processes and degradation of various molecular complexes was further intensified. Again, many of these biological regulations were attributed to the presence of the red complex. In conclusion, the present study provides the secreted proteome profile of gingival epithelial tissue to subgingival biofilms, and identifies a significant role for the red complex species in the observed effects. This article is protected by copyright. All rights reserved.