MicroRNAs (miRNAs) have emerged as critical regulators of diverse physiological processes, including skeletal remodeling. In our previous study, we identified miR-204–5p as significantly elevated in bone tissue during peak lactation, a physiological state characterised by transient osteoporosis due to low circulating estrogen levels. Given the similarity in hormonal changes observed during menopause, we investigated the putative role of miR-204–5p in regulating postmenopausal bone loss in this study. Functional assays demonstrated that miR-204–5p directly targets the 3′ untranslated region of Runx2 , a key transcription factor for osteoblast differentiation, and thus it recruits the target mRNA to the RNA-Induced Silencing Complex (RISC), as demonstrated in luciferase reporter and RNA immunoprecipitation (RNA-IP) assays. The miR-204–5p suppresses its identified target, Runx2, in a synergy of a combination of post-transcriptional mechanisms. Experimental evidence suggests that by enhancing the decay rate of the Runx2 mRNA and by impairing the protein turnover rate, the miRNA impedes its proposed target. Additionally, CRISPR/Cas9-mediated genetic manipulation of the studied miRNA further validated its regulatory role on osteoblast differentiation in vitro . In an ovariectomy-induced preclinical model of menopause, elevated miR-204–5p expression was found to be positively correlated with the bone loss, indicating its role in contributing to the pathophysiology of osteoporosis. Further studies confirm that the targeted inhibition of this miRNA restores the Runx2 expression and partially ameliorates the bone loss. Collectively, our findings establish miR-204–5p as a key modulator of estrogen-deficient bone loss and suggest that its inhibition may open more exciting and innovative strategies for osteoporosis prevention.