Abstract: Meiosis is regulated by phase-specific genes to orchestrate nuclear and cytoskeletal dynamics essential for sexual reproduction. The ciliate Paramecium tetraurelia exhibits nuclear dimorphism, harboring two germline micronuclei (MICs) and one somatic macronucleus (MAC) within a single cell during vegetative growth. During sexual reproduction, the MICs undergo meiosis and the MAC deforms and fragments, providing a unique model to study the regulation of diverse nuclear events. Transcription factor DP (TFDP), which heterodimerizes with E2Fs, can bind to specific DNA motifs in promoters of cell cycle-regulated genes to activate or repress their expression. Here, we identified 16 TFDP homologs in P. tetraurelia, representing an exceptional gene family expansion accompanied by functional domain diversification. The functions of Tfdp1a and Tfdp1b, which are specifically expressed during sexual reproduction and localize in the old and new MACs, were further investigated. Their depletion resulted in meiotic arrest at metaphase in the MIC, failure of old MAC fragmentation, and abortive cytokinesis. Transcriptomic analysis revealed that TFDP1A/1B knockdown primarily causes gene downregulation, with > 60% of downregulated genes being specifically highly expressed during sexual reproduction. Functional annotation and enrichment analyses demonstrated significant downregulation of proteins involved in meiosis, DNA replication, and DNA repair. Critically, multiple downregulated meiotic regulators are essential for proper homologous chromosome segregation and sister chromatid separation, providing a mechanistic basis for the observed MIC meiotic arrest. This study uncovers Tfdp1a/1b as essential regulators of the distinctive meiotic process in P. tetraurelia, providing crucial insights into nuclear dynamics and the regulation of sexual reproduction in binucleate systems.