To investigate the safety and efficacy of Self-Assembling Peptide P11-4 (SAP P11-4) compared to placebo or fluoride varnish (FV), a randomized, controlled, blinded, split-mouth study with sequential design was conducted. Subjects presenting two teeth with White-Spot-Lesions (WSLs) were included and teeth were randomly assigned to test or control. Control received placebo at baseline (D0) and test SAP P11-4, all received FV at Day 90 (D90). Standardized photographs were taken at each visit, and WSL size changes were morphometrically assessed. Hierarchical Linear Modelling, considering paired and sequential design, was used to test four hypotheses. SAP P11-4 lesions (test, D90-D0) showed significant WSL size reduction compared to placebo (control, D90-D0; p = 0.008) or FV (control, D180-D90; p = 0.001). Combination of SAP P11-4 and delayed FV after 90 days (test, D180-D0), showed a significant difference compared to FV alone (control D270-D90; p = 0.003). No significant difference on FV efficacy was found when SAP P11-4 was applied 3 months before FV (test D270-D90; control D270-D90, p = 0.70). SAP P11-4 treatment resulted in superior caries regression compared to either placebo or FV, and FV efficacy seems not to be affected by SAP P11-4. SAP P11-4 was found to be a safe and effective WSL treatment.