The contribution of ubiquitin-mediated mechanisms in the regulation of the Toxoplasma gondii cell cycle has remained largely unexplored. Here, we describe the functional characterization of a T. gondii deubiquitinase (TGGT1_258780) of the ovarian-tumor domain-containing (OTU) family, which, based on its structural homology to the human OTUD3 clade, has been designated TgOTUD3A. The TgOTUD3A protein is expressed in a cell cycle-dependent manner mimicking its mRNA expression, indicating that it is regulated primarily at the transcriptional level. TgOTUD3A, which was found in the cytoplasm at low levels in G1 parasites, increased in abundance with the progression of the cell cycle and exhibited partial localization to the developing daughter scaffolds during cytokinesis. Recombinant TgOTUD3A but not a catalytic-site mutant TgOTUD3A (C229A) exhibited activity against poly- but not monoubiquitinated targets. This activity was selective for polyubiquitin chains with preference for specific lysine linkages (K48 > K11 > K63). All three of these polyubiquitin linkage modifications were found to be present in Toxoplasma, where they exhibited differential levels and localization patterns in a cell cycle-dependent manner. TgOTUD3A removed ubiquitin from the K48- but not the K63-linked ubiquitinated T. gondii proteins independently of the modified target protein, thereby exhibiting the characteristics of an exodeubiquitinase. In addition to cell cycle association, the demonstration of multiple ubiquitin linkages together with the selective deubiquitinase activity of TgOTUD3A reveals an unappreciated level of complexity in the T. gondii "ubiquitin code." IMPORTANCE The role of ubiquitin-mediated processes in the regulation of the apicomplexan cell cycle is beginning to be elucidated. The recent analysis of the Toxoplasma "ubiquitome" highlights the importance of ubiquitination in the parasite cell cycle. The machinery regulating the ubiquitin dynamics in T. gondii has remained understudied. Here, we provide a biochemical characterization of an OTU (ovarian tumor) family deubiquitinase, TgOTUD3A, defining its localization and dynamic expression pattern at various stages of the cell cycle. We further establish that TgOTUD3A has activity preference for polyubiquitin chains with certain lysine linkages-such unique activity has not been previously reported in any apicomplexan. This is particularly important given the finding in this study that Toxoplasma gondii proteins are modified by diverse lysine-linked polyubiquitin chains and that these modifications are very dynamic across the cell cycle, pointing toward the sophistication of the "ubiquitin code" as a potential mechanism to regulate parasite biology.