TDP-43 proteinopathy, originally discovered in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), coexists with tauopathy in a variety of neurodegenerative disorders, including Alzheimer´s disease. However, the role of this co-occurrence in pathological processes is still unclear. In this study, we used transgenic SH-SY5Y-Tau-GFP cells, incubated with pre-formed Tau fibrils (Tau-PFF), to model and investigate the interaction between pathological Tau and endogenous TDP-43. We quantified TDP-43 intensity at cellular, nuclear and cytoplasmic levels using high-content single cell analysis. Tau-PFF treatment resulted in increased cellular, nuclear and cytoplasmic TDP-43 intensity. Interestingly, the nuclear/cytoplasmic (N/C) ratio decreased due to a larger increase in the cytoplasmic compartment, indicating abnormal subcellular redistribution as observed in TDP-43 proteinopathies. We also analyzed the effect of Tau-PFF incubation in cell populations with (+) or without (-) stable Tau-GFP expression. Nuclear TDP-43 intensity increased upon Tau-PFF treatment regardless of Tau-GFP expression. Moreover, we detected an increase in nuclear TDP-43 intensity when comparing Tau-GFP (+) versus (–) cells within both control and Tau-PFF treated groups, with the largest fold-change showed by the control Tau-GFP (-) vs. Tau-PFF Tau (+) comparison. In summary, this data demonstrate a causal role for pathological, fibrillar Tau in regulating TDP-43 levels and distribution.