Background: The aim of the study was to analyze the effect of Dimethylenaston, a mitotic kinesin 5 (Eg5) inhibitor, in an experimental setting of glaucoma filtration surgery. Methods: On 37 chinchilla rabbits (ChBBCH), glaucoma filtration surgery similar to clinical practice, was performed. The animals received either no adjuvant, one unilateral subconjunctival injection of Dimethylenastron (1.0 mol, 3.0 mol), or the vehicle alone at baseline and in two further groups additionally at days 3 and 7 thereafter (1.0 mol, 3.0 mol). The evaluation of antifibrotic efficacy was performed by clinical response, histological examination, and immunohistochemical staining for smooth muscle actin (SMA) and CD 31. The animals were sacrificed on day 14, and the eyes processed for histology. Results: The vehicle was well tolerated. Except for two cases of transient fibrinous reaction after the injection of 3.0 mol Dimethylenastron, no adverse effects, such as inflammation or blurring of the optical media, were observed. A bleb scarring occurred in the group that received surgery only, adjuvant DMSO, or Dimethylenastron 3.0 mol. Dimethylenastron (1.0 mol) induced a milder scarring compared with the control group but the length of bleb survival was not significantly prolonged (p0.053, Kaplan-Meier log rank test). In all groups, the intraocular pressure correlated with the fibrotic process and reached normal levels within 14 days after surgery. Those groups injected with 1.0 mol Dimethylenastron revealed a significantly reduced ratio of intraocular pressure and a milder, but not sufficiently reduced, subconjunctival fibrotic reaction according to the histological and immunohistochemical analysis. Conclusions: The subconjunctival administration of Dimethylenastron 1.0 mol induced a milder conjunctival scarring. The applied concentrations of Dimethylenastron did not improve the surgical outcome of glaucoma filtration treatments in rabbits sufficiently.