Circular RNA (CircRNA) plays a potential role in bone formation. We aimed to study the circRNAs expression profiles and their functions in osteogenic differentiation of human bone marrow stromal cells (BMSCs). Firstly, we established osteogenic differentiation of BMSCs displaying increased mRNA expression of osteogenic differentiation marker (RUNX2, OPN, and OCN), increased ALP activity and protein expression, and increased mineralized nodules formation, as well as morphological alteration. Then, we employed high-throughput sequencing to analyze circRNA expression and found that 3440 and 3893 circRNAs in non-induced and induced groups, respectively. We further validated the 10 differentially expressed circRNAs with the most significant difference between induced and non-induced groups. Among these ten circRNAs, five of them with more than one miRNA binding site were used to construct a ceRNA network exhibiting 81 miRNAs and 182 target mRNAs. Furthermore, among these five circRNAs, we found only circ_0005564 significantly reduced the mRNA expression of RUNX2, OPN, and OCN. The circularity of circ_0005564 was verified. Our results showed that knockdown of circ_0005564 inhibited osteoblast differentiation in BMSCs. Taken together, our study demonstrates that circ_0005564 is a potential positive regulator of osteogenic differentiation of BMSCs.