Background: Mesenchymal stromal cells (MSCs) have high differentiation potential into different cell lines like osteoblasts. Osteogenic differentiation can be regulated through various and complex mechanisms, such as changing the microRNAs expression level. Although platelet-rich plasma (PRP) has been used in MSCs’ osteogenic differentiation process, the molecular mechanisms underlying the effect of PRP induction of osteogenesis by microRNAs is not well understood. Objectives: We evaluated the effect of PRP on the expression of miR-29a, miR-29b, and miR-155 in the PRP-based osteogenic differentiation of human MSCs. Methods: This experimental study was conducted on healthy individuals referred to Taleghani Hospital in Ahvaz, Iran, for abdominoplasty from September 2017 to April 2018. Stromal cells were isolated from human adipose tissue and differentiated into osteoblasts. Effect of 10% PRP on osteoblasts differentiation was monitored by the measurement of alkaline phosphatase activity and calcium deposition. We also evaluated gene expression of the Runx2 and the OPN along with the expression of miRNAs. All tests were performed in triplicate. Results: Treatment of MSCs with 10% PRP resulted in induction of osteogenic differentiation by a significant upregulation of the miR-29a/b (miR-29a: 5.27 (0.77), P < 0.01 (day 3) and 3.76 (0.124), P < 0.01 (day 14); miR-29b: 3.11 (0.35), P < 0.001 (day 3) and 4.25 (0.304), P < 0.01 (day 14)) and a significant downregulation in the miR-155 expression (0.62 (0.006), P < 0.001 (day 3) and 0.55 (0.114), P < 0.05 (day 14)). Conclusions: The remarkable rise in the expression of important osteoblast genes, alkaline phosphatase activity, and calcium deposition verified accelerated differentiation. The present study showed that microRNAs such as miR-29a/b and miR-155 play an active role in the process of bone differentiation during PRP treatment, which in turn, affects mesenchymal cells signaling pathways.