Osteosarcoma (OS), one of the bone tumors, occurs mainly during childhood and adolescence and has an incidence rate of 5%. Cinnamtannin B-1 (CTB-1) is a natural trimeric proanthocyanidin compound found in plants Cinnamomum zeylanicum and Laurus nobilis. Previously, several articles have demonstrated that CTB-1 exerts a certain effect on melanoma and cervical cancer. However, their role in OS remains unclear. In this study, CTB-1 was found to inhibit the proliferation of OS cancer cells, with the dose of CTB-1 positively correlated to the survival rate of HOS and MG-63 cells. Recently, microRNAs (miRNAs) were also reported to play an important role in tumor proliferation. Hence, we performed the miRNA sequencing analysis after CTB-1 treatment to identify miRNA levels in HOS cells and found that the expression of miR-1281 was significantly upregulated. According to the functional analysis, CTB-1 inhibited the growth and migration of OS by upregulating the expression of miR-1281. Additionally, miR-1281 acted as a sponge for Peptidylprolyl Isomerase F (PPIF), inhibiting its expression levels. The rescue experiments revealed that CTB-1 delayed the development of OS by regulating the miR-1281/PPIF pathway. Hence, our findings suggested that CTB-1 inhibited the cell growth, invasion, and migration of OS by upregulating miR-1281 and inhibiting PPIF expression, thereby providing a possible target drug for OS treatment.