We have shown that Benznidazole (BZL), a compound with well documented trypanocidal activity, possesses anti-inflammatory properties and inhibits the nuclear factor κB (NF-κB). Given the relationship between this transcription factor and cell growth, in this study we address the role of NF-κB blockade by BZL in the proliferation of different cell lines. Our studies demonstrate that this compound significantly reduced proliferation of RAW 264.7 macrophage cell line, as assessed by trypan blue exclusion, MTT reduction and [3H]-thymidine incorporation, at a concentration shown to inhibit NF-κB. Treatment with BZL also led to growth arrest in CHO, MDCK and HeLa cells. Interestingly, growth inhibition was found to be a reversible process, not accompanied by significant cell death, indicating that the drug behaves mainly as a cytostatic compound. As this effect might be related to NF-κB inhibition, we next evaluated whether other NF-κB inhibitors could induce growth arrest in RAW 264.7 and HeLa cells. We found that IKK inhibition led to growth arrest in both cell lines, indicating that NF-κB inhibition may be the potential mechanism by which BZL inhibits cell proliferation. To the best of our knowledge, this is the first report of an anti-proliferative activity of the trypanocidal drug against different cell lines and provides a mechanistic insight that may help understand some of the adverse effects associated with prolonged treatment.