Keratoconus (KC) is the most common primary corneal ectatic disease which has considerable importance in public health. Corneal collagen crosslinking (CXL) is a procedure to mitigate progression of KC and reduce demand for corneal transplantation. Although studies have proven the efficacy of CXL regarding corneal shape, none have investigated the effects of CXL on tear biomarkers which are useful tools to understand molecular mechanisms behind CXL. Our purpose was to determine the effect of CXL on tear mediators in patients with KC and analyze associations with corneal changes. Tear samples were collected pre-CXL from 26 eyes of 23 patients and during a 12-month follow-up. The mediators' concentration was measured by Cytometric Bead Array technology. Corneal topography parameters measured by Scheimpflug Camera included: Thinnest-corneal-thickness (ThCT), keratometry values (K1, K2), Radii-Minimum (Rmin), Keratoconus-Index (KI), Center-KI (CKI), Index-of-Height Asymmetry (IHA) and Index-of-Surface Variance (ISV). At baseline, KI was correlated negatively with chemokine (C-C motif) ligand 5 (CCL5) (p=0.015) and matrix metalloproteinase (MMP)-13 (p=0.007). At day 4, interleukin (IL)-6 and IL-8 increased, while IL-13, IL-17A, interferon (IFN)-γ, CCL5, MMP-13, epidermal growth factor (EGF), nerve growth factor (NGF) and plasminogen activator inhibitor (PAI-1) decreased significantly compared to pre-CXL concentrations (p≤0.02). At 6 months tissue plasminogen activator (t-PA) increased (p=0.02), while at 12 months Rmin increased (p≤0.004), and IL-6 and CXCL8 (p=0.005 and p=0.047) as well as K1, ISV and KI decreased. After 6 months CKI and ISV showed significant associations with IL-17A; CKI with IL-13 and ThCT with IL-13 (p≤0.02), while at 12 months there were reverse associations between ThCT and IL-6, IL-13, INFγ, CCL5 and PAI-1 (p≤0.02). Alterations of mediators in tear fluid after CXL associate with topographic changes highlight the fact that many mediators are involved in the complex mechanisms after CXL. Further studies on biomarkers to investigate the efficacy of CXL are needed.