The focal adhesion protein vinculin (VCL) is a cytoskeletal protein associated with cell-matrix junctions where it acts as a molecular clutch between the actin cytoskeleton and the extracellular matrix via talins and integrins. However, our study reveals new localization and unexpected role of VCL in the nuclei of spermatocytes during Prophase I. In particular, VCL localizes in the nuclear interior along the meiosis-specific structure synaptonemal complex (SC) and in the centromeric regions of homologous chromosomes. To understand the role of VCL in the early meiotic division, we prepared a VCL conditional knock-out mouse model (VCL cKO) where the VCL deletion is manifested explicitly during the early stages of spermatogenesis. In comparison with the wild-type control, our mouse model shows significantly decreased ability of males to sire offspring together with increased percentage of malformed sperm. According to our short-term cultivation experiments primary spermatocytes of VCL cKO males exhibit deficiency in progression throughout late prophase I. Likewise, FACS (fluorescence-activated cell sorting) analysis revealed the enrichment of the prophase I spermatocytes. The chromosomal pairing and the crossing-over formation appear intact. However, spermatocytes in late prophase I show a premature separation of homologous chromosomes with partial loss of the central element of SC and significantly increased distance between the centromeres. Taken together, our findings indicate that VCL plays an important role in the meiotic progression. We show that the VCL deletion in spermatocytes leads to considerable abnormalities on the level of chromosomal desynapsis which in the end results in the decreased breeding capability of VCL cKO males. Funding: This study is supported by the CSMS scholarship, GACR (16-03403S), the project „BIOCEV“ (CZ.1.05/1.1.00/02.0109) and Light Microscopy Core Facility, IMG ASCR.