Cell cycle progression in mammals is strictly controlled by a number of cyclin-dependent kinases (CDKs) and CDK inhibitors (CKIs), the expression of which is often dysregulated in cancer cells. Our previous work revealed that Cullin 4B (CUL4B), a critical component of the Cullin4B-RING E3 ligase complex (CRL4B), is overexpressed in human osteosarcoma cells through an unknown mechanism. Here, we demonstrated that CUL4B forms an E3 ligase with RBX1 (RING-box 1), DDB1 (DNA damage binding protein 1), and DCAF11 (DDB1 and CUL4 associated factor 11) in human osteosarcoma cells. In vitro and in vivo ubiquitination analyses indicated that CRL4BDCAF11 E3 ligase was able to specifically ubiquitinate a CDK inhibitor-p21Cip1 at K16, K154, K161 and K163 but not at K75 and K141. Knocking down any component of the CRL4BDCAF11 complex, including CUL4B, DDB1 or DCAF11, using short hairpin RNAs (shRNAs) attenuated the ubiquitination level of p21Cip1, inhibited osteosarcoma cell proliferation, led to cell cycle arrest at S phase, and decreased colony formation rate. Taken together, our data suggest that the CRL4BDCAF11 complex represents a unique E3 ligase that promotes the ubiquitination of p21Cip1 and regulates cell cycle progression in human osteosarcoma cells.