The mechanism that regulates sperm release at spermiation is unknown. Herein, we utilized an animal model wherein rats were treated with adjudin, 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide, via oral gavage to induce premature release of elongating/elongated spermatid, followed by round spermatids and spermatocytes. Spermatid release mimicking spermiation occurred within 6-12 h following adjudin treatment and by 96 h, virtually all tubules were devoid of spermatids. Using this model, we tracked the organization of F-actin and microtubules (MTs) by immunofluorescence microscopy, and the association of actin or MT regulatory proteins that either promote or demolish cytoskeletal integrity through changes in the organization of actin-microfilaments or MTs by co-immunoprecipitation. Adjudin treatment induced an increase in the association of: (i) Eps8 (an actin barbed-end capping and bundling protein) or formin 1 (an actin nucleator) with actin, and (ii) EB1 (a MT stabilizing protein) with MT shortly after adjudin exposure (at 6 h), in an attempt to maintain spermatid adhesion to the Sertoli cell at the apical ectoplasmic specialization (apical ES). However, this is followed by a considerable decline of their steady-state protein levels, replacing with an increase in association of (i) Arp3 (a branched actin nucleator that converts actin filaments into a branched/unbundled network) with actin, and (ii) MARK4 (a MT destabilizing protein kinase) with MTs by 12 h after adjudin treatment. These latter changes thus promote actin and MT dis-organization, leading to apical ES disruption and the release of sperm, mimicking spermiation. In summary, spermiation is a cytoskeletal-dependent event, involving regulatory proteins that modify cytoskeletal organization.