SAG/RBX2 and RBX1 are two family members of RING components of Cullin-RING ligases (CRLs), required for their enzymatic activity. Previous studies showed that SAG prefers to bind with CUL5, as well as CUL1, whereas RBX1 binds exclusively to CULs1–4. Detailed biochemical difference between SAG and RBX1, and whether SAG mediates cross-talk between CRL5 and CRL1 are previously unknown. Here we report that the levels of SAG and β-TrCP1 are inversely correlated, and SAG-CUL5-βTrCP1 forms a complex under physiological condition. SAG-CUL5, but not RBX1-CUL1, negatively modulates β-TrCP1 levels by shortening its protein half-life through promoting its ubiquitylation via atypical K11-linkage. Consistently, chemical inducers of SAG reduced β-TrCP1 level. Furthermore, SAG mainly binds to E2s UBCH10 and UBE2S known to mediate K11 linkage of ubiquitin, whereas RBX1 exclusively binds to E2s CDC34 and UBCH5C, known to mediate K48 linkage of ubiquitin. Finally, silencing of either UBCH10 or UBE2S, but not UBCH5C, caused accumulation of endogenous β-TrCP1, suggesting that β-TrCP1 is a physiological substrate of SAG-UBCH10C/UBE2S. Our study, for the first time, differentiates SAG and RBX1 biochemically via their respective binding to different E2s; and shows a negative cross-talk between CRL5 and CRL1 through SAG mediated ubiquitylation of β-TrCP1.