Objectives The aim of this study was to assess the concentrations of heat shock protein 70 (HSP70) and toll-like receptor 4 (TLR4) during orthodontic tooth movement and to compare their levels with interleukin-1 beta (IL-1 beta), a well-known proinflammatory biomarker. Materials and methods This study consisted of 20 patients (8 males, 12 females; mean age 14.75 +/- 2.34 years) who needed maxillary premolar extraction and segmental canine distalization. Concentrations of HSP70, TLR4, and IL-1 beta were examined before extraction (T1), at the 1st (T2), 4th (T3), 7th (T4), 14th (T5), and 30th (T6) days of canine retraction by enzyme-linked immunosorbent assay analysis of gingival crevicular fluid samples. Statistical analyses were performed with repeated measure ANOVA and Spearman's rank correlation coefficient analysis (p < 0.05). Results HSP70 increased gradually from T1 to T6 and showed significant differences between T1-T6 and T2-T6 (T1:3.28 +/- 0.92 ng/ml; T2:3.72 +/- 0.66 ng/ml; T6:9.35 +/- 2.45 ng/ml). The lowest TLR4 concentration was at T1, peaked at T3 and remained constant afterwards with significant differences between T1-T3, T1-T4, and T1-T6 (T1:0.71 +/- 0.02 pg/ml; T3:1.04 +/- 0.11 pg/ml; T4:0.95 +/- 0.06 pg/ml; T6:1.00 +/- 0.07 pg/ml). IL-1 beta increased from T1 to T6 with significant differences between T1-T4, T1-T5, and T1-T6 (T1:55.71 +/- 5.48 pg/ml; T4:100.11 +/- 16.92 pg/ml; T5:103.71 +/- 23.19 pg/ml; T6:125.12 +/- 22.04 pg/ml). The increase in HSP70 and TLR4 from T2-T3 showed a significant correlation (r = 0.598; p = 0.005). Conclusions The increased levels of HSP70, TLR4, and IL-1 beta show the contribution of these mediators to the inflammatory response from the early stages of orthodontic tooth movement.