Elimination of protein expression using RNA interference (RNAi) significantly improves the understanding of gene function and represents a promising technique for the treatment of diseases such as cancer and neurological disorders. Accumulating evidence suggests the so-called interferon-independent non-specific gene silencing of short interfering RNA (siRNA); however, its biological and functional cellular consequences are largely unidentified. We therefore analyzed the effects of different nonsense siRNAs on characteristic bio-parameters such as cell viability, proliferation, cell cycle distribution, apoptosis, and migration of tumor cells. All analyzed cellular aspects have been observed to be significantly affected by the presence of siRNA in an interferon-independent manner: viability, mitosis, and motility were significantly diminished and programmed cell death was significantly elevated. Moreover, all cell cycle stages (G0/G1-, G2/M-, and S-phase) were moderately shifted. Together, these results support the hypothesis that siRNA, due to sequence-specific cellular consequences, modulate bio-functionality independent of the target sequence. This phenomenon affects the design of siRNA experiments for future in vitro but also for in vivo tests as well as for potential therapeutic and preventive strategies. Moreover, monitoring interferon response after transfection of siRNAs is necessary but not sufficient to exclude potential off-target effects in non-diseased cells.