Alpha4 (alpha4) is a key regulator of protein phosphatase 2A (PP2A) and mTOR in steps essential for cell-cycle progression. alpha4 forms a complex with PP2A and MID1, a microtubule-associated ubiquitin E3 ligase that facilitates MID1-dependent regulation of PP2A and the dephosphorylation of MID1 by PP2A. Ectopic overexpression of alpha4 is associated with hepatocellular carcinomas, breast cancer, and invasive adenocarcinomas. Here, we provide data suggesting that alpha4 is regulated by ubiquitin-dependent degradation mediated by MID1. In cells stably expressing a dominant-negative form of MID1, significantly elevated levels of alpha4 were observed. Treatment of cells with the specific proteasome inhibitor, lactacystin, resulted in a 3-fold increase in alpha4 in control cells and a similar level in mutant cells. Using in vitro assays, individual MID1 E3 domains facilitated monoubiquitination of alpha4, whereas full-length MID1 as well as RING-Bbox1 and RING-Bbox1-Bbox2 constructs catalyzed its polyubiquitination. In a novel non-biased functional screen, we identified a leucine to glutamine substitution at position 146 within Bbox1 that abolished MID1-alpha4 interaction and the subsequent polyubiquitination of alpha4, indicating that direct binding to Bbox1 was necessary for the polyubiquitination of alpha4. The mutant had little impact on the RING E3 ligase functionality of MID1. Mass spectrometry data confirmed Western blot analysis that ubiquitination of alpha4 occurs only within the last 105 amino acids. These novel findings identify a new role for MID1 and a mechanism of regulation of alpha4 that is likely to impact the stability and activity level of PP2Ac.