Objective: In the heart of humans and other mammals, low renewal capacity is unable to restore heart function after any significant injury. Recent studies have shown that some microRNAs (miRNAs) inhibit cardiomyocytes proliferation by repressing the expression of multiple cell cycle regulators. Some of well-known cardiac muscle transcriptional networks, consisting of SRF/ myocardin are responsible for the expression of these miRNA genes. In according to other literatures, miR-1 -2/133a-1 and miR-206/133b are two of these miRNA clusters which are expressed in cardiac and skeletal muscle, respectively. In contrast to other studies, we showed the differential expression levels of hsa-miR-133b during cardiac differentiation. Also, miR-133b differs from miR-133a by only two nucleotides at the 3’terminus. The sequence similarity of these miRNAs suggests that they share the same or similar regulatory mRNA targets. These findings could award us to suppose the potential roles for miR-133b in cardiovascular system, as well.Materials and Methods: The human cardiac progenitor cells (c-Kit+progenitor cells) were prepared from Royan Stem Cell Bank (RSCB), cultured and differentiated into cardiomyocytes. The differentiation into cardiomyocytes was confirmed by ICC (Immunocytochemistry) test for cardiac troponinT and real-time PCR for some early cardiac marker and sarcomeric genes. The expression profiles of hsa-miR-133b and some of its target genes including of SRF, CCND2, TGFBR1, TGFBR2 and TGFBR3 were analyzed during the processes of differentiation.Results: Real time PCR data showed the increasing levels of hsa-miR-133b during cardiac differentiation stages. The expression levels of this miRNA in 3-4 weeks after the first differentiation induction was about five times higher than that of early stage (p<0.05). In terminal differentiated cardiomyocytes, the transcriptional level of this miRNA was decreased (p<0.05). In addition, the expression profiling of upper mentioned target genes during cardiomyocytes differentiation showed the reverse pattern of hsa-miR-133b expression profile.Conclusion: In the heart, SRF binds the enhancer regions of the miR-1/miR-133 cluster and regulates their expression in cardiomyocytes. In this view, over-expression of miR-133b might blocks cardiomyocytes proliferation and differentiation with direct targeting of SRF (in a negative feed-back loop), CCND2, TGFBR1, TGFBR2 and TGFBR3. Altogether, our data award us into the regulatory networks of related genes that are involved in cardiomyocytes differentiation and proliferation.