Objective: To investigate the effect of miR-27b-3p on osteosarcoma cell growth and its potential molecular mechanism. Methods: ①The expression of miR-27b-3p in osteosarcoma cell lines was analyzed by qRT-PCR. ②CCK-8 assay, colony formation assay and flow cytometry analysis were used to detect the effect of miR-27b-3p and SSRP1 on osteosarcoma cell growth.③The miRNA target prediction database was used to predicted potential target gene of miR-27b-3p, and dual luciferase reporter assays were performed to demonstrate the relationship of miR-27b-3p and its target genes. ④Efficiency of knockdown or overexpression was validated by Western blot analysis. Results: ①miR-27b-3p was lowly expressed in osteosarcoma cell lines and clinical samples, and overexpression of miR-27b-3p led to suppress OS cell growth. ② Bioinformatics analyses and dual-fluorescence reporter assays confirmed that SSRP1 was a target gene of miR-27b-3p, and the expression of SSRP1 can be regulated by miR-27b-3p. ③Silencing of SSRP1 significantly inhibited cell proliferation and increased cell apoptosis in OS. ④Overexpression of SSRP1 partly reversed the inhibitory effect of miR-27b-3p on OS cell growth. Conclusion: miR-27b-3p was downregulated and regulated OS cell growth by targeting SSRP1, this study confirmed that miR-27b-3p was a potential therapeutic target in OS and targeting the miR-27b-3p/SSRP1 axis may become a new therapeutic strategy for the treatment of OS.