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Cytoplasmic microtubules are tubular polymers that can harbor small proteins or filaments inside their lumen. The identities of these objects and mechanisms for their accumulation have not been conclusively established. Here, we used cryogenic electron tomography of Drosophila S2 cell protrusions and found filaments inside the microtubule lumen, which resemble those reported recently in human HAP1 cells. The frequency of these filaments increased upon inhibition of the sarco/endoplasmic reticulum Ca2+ ATPase with the small molecule drug thapsigargin. Subtomogram averaging showed that the luminal filaments adopt a helical structure reminiscent of cofilin‐bound actin (cofilactin). Consistent with this, we observed cofilin dephosphorylation, an activating modification, in cells under the same conditions that increased luminal filament occurrence. Furthermore, RNA interference knock‐down of cofilin reduced the frequency of luminal filaments with cofilactin morphology. These results suggest that cofilin activation stimulates its accumulation on actin filaments inside the microtubule lumen. Synopsis: This cryogenic electron tomography (cryoET) study shows the presence of filaments inside the microtubule lumen of induced cellular protrusions of Drosophila S2 cells. The luminal filaments accumulate in response to specific cell stress and the majority are formed of cofilin‐bound actin (cofilactin). CryoET shows that microtubules inside induced protrusions of Drosophila S2 cells can contain filaments in their lumen.Treatment with the SERCA inhibitor thapsigargin increases the frequency of luminal filaments.Subtomogram averaging and cofilin knock‐down experiments suggest that the luminal filaments are formed of cofilin‐bound actin (cofilactin). [ABSTRACT FROM AUTHOR]