Male-typical behaviors such as aggression and mating, which reflect sexual libido in male mice, are regulated by the hypothalamus, a crucial part of the nervous system. Previous studies have demonstrated that microRNAs (miRNAs), especially miR-29, play a vital role in reproduction and the neural control of behaviors. However, it remains unclear whether miR-29 affects reproduction through the hypothalamus-mediated regulation of male-typical behaviors. Here, we constructed two mouse knockout models by ablating either the miR-29ab1 or miR-29b2c cluster. Compared to WT, the ablation of miR-29ab1 in male mice significantly reduced the incidence of aggression by 60% and the incidence of mating by 46.15%. Furthermore, the loss of miR-29ab1 in male mice led to the downregulation of androgen receptor (AR) in the ventromedial hypothalamus. Transcriptomic analysis of the hypothalamus of miR-29ab1-deficient mice revealed inflammatory activation and aberrant expression of genes associated with male-typical behaviors, including Ar, Pgr, Htr4, and Htr2c. Using bioinformatics analysis and dual-luciferase reporter assays, we identified zinc finger protein 36 (Zfp36) as a direct downstream target gene of miR-29ab1. We subsequently showed that ZFP36 colocalized with AR in GT1-7 cells. Furthermore, inhibition of Zfp36 or RelB in GT1-7 cells led to an increase in AR expression. Collectively, our results demonstrate that the miR-29ab1/Zfp36/AR axis in the hypothalamus plays a pivotal role in the regulation of aggression and mating in male mice, providing a potential therapeutic target for treating infertility caused by low libido.