Class III beta-tubulin (TUBB3) overexpression has been reported in ovary, lung, breast, and gastric cancer patients. Currently, no clinical drugs are available for a specific targeting of TUBB3, whereas the investigational drug IDN5390 specifically interacts with TUBB3. To gain insight into the pathways leading to TUBB3 up-regulation, we did a human genome microarray analysis in A2780 cells made resistant to IDN5390 to identify selected pathways specifically disrupted in resistant cells. Using this approach, we discovered that semaphorin 6A (SEMA6A) is down-regulated not only in IDN5390-resistant cells but also in cells made resistant to cisplatin, topotecan, and doxorubicin, whereas no changes were noticed in paclitaxel- and gemcitabine-resistant cells. Acute treatment with IDN5390 was able to down-regulate SEMA6A in cells unselected for drug resistance. TUBB3 expression was assessed in A2780 clones with stable overexpression of SEMA6A and in a panel of clones in which silencing of the protein was obtained. Quantitative PCR was then used to check the modulation of SEMA6A as well as to assess the expression of TUBB3. TUBB3 was increased (median value, 5.4) and reduced (median value, 0.47) in cells with overexpression and silencing of SEMA6A, respectively. Thus, the findings indicate a correlation between the expression of SEMA6A and TUBB3. Then, we found that a form of 83 kDa of SEMA6A is expressed in the cytoskeleton in association with beta-actin. These findings suggest for SEMA6A a novel function in the cytoskeleton and a role in modulating tubulin isotype composition and microtubule dynamics.