Abstract: Objective To investigate the effect of short hairpin RNA （shRNA） targeting protein kinase Cδ（PKCδ） on the proliferation， apoptosis and related signaling pathways of human osteosarcoma stem cells. Methods According to the PKCδ sequence in Genbank database， the specified shRNA series for PKCδ （PKCδ-shRNA-1 and PKCδ-shRNA-2） were designed， synthesized， and then cloned into pRNA6-1-Neo plasmid vector. The Western blotting was used to detect the level of PKCδ at 24， 48， 72， and 96 h after transfectioin. The shRNA with the best inhibition effect was chosen to transfect osteosarcoma stem cells （Transfection group）， and the stem cells transfected with the negative control sequence （Negative control group） and without any transfection （Blank control group） were prepared. The proliferation inhibition rates were measured by Thiazolyl blue （MTT） at 24， 48， 72， and 96 h after transfectioin. The flow cytometry was used to measure the apoptosis and cell cycle. Fluorescent quantitative PCR was used to detect the expression of related apoptotic genes. Western blotting was used to detect the levels of related protein in PI3K／Akt and Wnt／β-catenin pathways at 96 h after transfection. Results The level of PKCδ decreased after the transfection of PKCδshRNA1 and PKCδshRNA2. Given the better inhibition effect of PKCδ-shRNA-1， PKC-shRNA-1 was used in the subsequent experiment. Compared with other two groups， the proliferation inhibition rate， apoptosis rate and mRNA levels of Bax and Bad were increased， while the Bcl-2 level was decreased in Transfection group （P＜0.05）. The proportion of G0／G1 phase was increased， but the S phase and G2／M phase cells were decreased in Transfection group versus other groups （P＜0.05）. In PI3K／Akt pathway， the level of PTEN was increased， but the level of Akt was decreased. In Wnt／β-catenin pathway， the levels of β-catenin， C-myc and Cyclin D1 were decreased in the transfected group（P＜0.05）. Conclusion ShRNA inhibits the proliferation and induces apoptosis and cell cycle arrest of osteosarcoma stem cells by inhibiting the expression of PKCδ gene.