in response to mechanical and biochemical cues, allowing the cell to rapidly tune its mechanics from highly-crosslinked, elastic networks to weaklycrosslinked viscous networks. While the role of actin crosslinking in controlling actin network mechanics is clear in purified actin networks, its mechanical role in the cytoplasm of living cells remains unknown. Here, we probe the frequency-dependent viscoelastic properties of the cytoplasm in living cells using multiharmonic excitation and in situ optical trap calibration. At previously unexplored long timescales, we observe that the cytoskeleton fluidizes. The mechanics are well-captured by a model in which actin filaments are dynamically connected by a single dominant crosslinker. A disease-causing point mutation (K255E) of the actin crosslinker a-actinin 4 (ACTN4) causes its binding kinetics to be insensitive to tension. Under normal conditions, the viscoelastic properties of wild type (WT) and K255Eþ/-cells are similar. However, when tension is reduced through myosin II inhibition, WT cells relax 3x faster to the fluid-like regime while K255Eþ/-cells are not affected. These results indicate that dynamic actin crosslinking enables the cytoplasm to flow at long timescales.