Alfalfa ( Medicago sativa L.) is a high-quality forage crop and an essential resource for livestock. Understanding the molecular mechanisms underlying male sterility in alfalfa is pivotal for the development of superior forage varieties. Despite the critical role of anther development in plant reproduction, its molecular regulation—particularly the involvement of transcription factors in M. sativa—remains insufficiently explored. This study bridges this gap by isolating and characterizing an R2R3-MYB transcription factor, MsMYB35, and unveiling its regulatory role in anther development. Quantitative RT-PCR (qRT-PCR) revealed that MsMYB35 is predominantly expressed during early anther development and is homologous to AtMYB35. MsMYB35 was found to localize in both the cytoplasm and nucleus. DNA affinity purification sequencing (DAP-seq) identified 3,647 target genes of MsMYB35, with enrichment analysis uncovering three recognition motifs. Integrated DAP-seq and RNA-seq analyses revealed that MsMYB35 directly regulates two key anther development-related genes. Functional analyses showed that overexpression of MsMYB35 promotes anther development, while silencing MsMYB35 leads to defective anther sacs and wrinkled pollen grains. Proper MsMYB35 expression ensures the formation of viable and fertile pollen grains, solidifying its role as a critical regulator of anther development. These findings provide a novel perspective on the molecular mechanisms regulating anther development in M. sativa and offer valuable insights for improving molecular breeding and hybrid seed production strategies. By advancing the fundamental understanding of transcriptional regulation in anther development, this study sets the stage for innovative approaches to alfalfa crop improvement.