Background Adipogenesis is a highly organised series of events that facilitates the healthy expansion of adipose tissue, beginning during embryogenesis and continuing throughout life. White adipogenesis protects against lipotoxicity, influencing insulin resistance and obesity-related comorbidities. Brown adipogenesis enhances energy expenditure, thereby counteracting weight gain, lipotoxicity and insulin resistance. Recently, there has been a significant increase in interest regarding adipocyte differentiation, mainly focusing on the interplay between microRNAs (miRNAs) and the transcriptional cascade that governs adipogenesis and metabolic dysfunction. This study aimed to identify miRNAs regulating white and brown adipocyte differentiation and define miRNA action in a stem cell model of adipogenesis. Methods Small RNAseq analysis of primary mouse brown and white adipocytes (WAs) identified miR-10b to be upregulated in mature brown adipocytes (BAs). We generated two model systems: 1) immortalized brown pre-adipocytes treated with miRNA inhibitors and 2) CRISPR/Cas9 KO of miR-10b in E14 mouse embryonic stem cells (mESCs). Both cell models were differentiated into mature adipocytes. To unravel the pathways that are affected by miR-10b depletion, a transcriptomic analysis was performed at key time points. Results Both cell models showed that miR-10b-5p depletion severely impaired differentiation into mature adipocytes, as indicated by a lack of lipid droplet formation and reduced adipogenic gene expression. Gene expression analysis supports that miR-10b-5p directs embryonic stem (ES) cells towards the mesoderm lineage, promoting commitment to pre-adipocytes by downregulating Gata6 and its downstream target Bmp2. This mechanism appears to be unaffected in BAs. Our study demonstrated that miR-10b-5p regulates the later stages of adipogenesis, at least in part, by downregulating Tub, a direct target of miR-10b-5p. We also confirmed that miR-10b-5p alleviated the halted differentiation phenotypes of adipocytes by supressing the G Protein Signalling pathway mediated by Tubby. Conclusions These results evidence that miR-10b inhibition plays a dynamic role in adipocyte biology, as its inhibitory effects manifest differently during the stem cell preadipocyte proliferation state and during the maturation phase of adipocytes. Collectively, our study demonstrated that miR-10b-5p may represent a new potential therapeutic target for lipodystrophy and obesity.