Ubiquilin-1 is an Alzheimer's disease-associated protein, which is known to modulate amyloid precursor protein (APP) processing, amyloid-β (Aβ) secretion, and presenilin-1 (PS1) accumulation. Here, we aim to elucidate the molecular mechanisms by which full-length transcript variant 1 of ubiquilin-1 (TV1) affects APP processing and γ-secretase function in human neuroblastoma cells stably overexpressing APP (SH-SY5Y-APP751). We found that TV1 overexpression significantly increased the level of APP intracellular domain (AICD) generation. However, there was no increase in the levels of secreted Aβ40, Aβ42, or total Aβ, suggesting that ubiquilin-1 in particular enhances γ-secretase-mediated ε-site cleavage. This is supported by the finding that TV1 also significantly increased the level of intracellular domain generation of another γ-secretase substrate, leukocyte common antigen-related (LAR) phosphatase. However, in these cells, the increase in AICD levels was abolished, suggesting a preference of the γ-secretase for LAR over APP. TV2, another ubiquilin-1 variant that lacks the protein fragment encoded by exon 8, did not increase the level of AICD generation like TV1 did. The subcellular and plasma membrane localization of APP or γ-secretase complex components PS1 and nicastrin was not altered in TV1-overexpressing cells. Moreover, the effects of TV1 were not mediated by altered expression or APP binding of FE65, an adaptor protein thought to regulate AICD generation and stability. These data suggest that ubiquilin-1 modulates γ-secretase-mediated ε-site cleavage and thus may play a role in regulating γ-secretase cleavage of various substrates.