Morpholino phosphorodiamidate antisense oligonucleotides (MOs) and short interfering RNAs (siRNAs) are commonly used to study gene function through sequence-specific knockdown. However, both technologies can cause off-target effects. This study shows that MOs induce off-target effects through p53 activation, leading to cell death. The major off-target effect is mediated by p53 activation, as detected by TUNEL assay, acridine orange, and p21 transcriptional activation. Concurrent knockdown of p53 ameliorates the cell death caused by MO off-targeting. Interestingly, p53 knockdown does not affect specific gene function loss, such as the cell death caused by loss of function of chordin. MO off-target effects are accompanied by diagnostic transcription of an N-terminal truncated p53 isoform. This study shows that MO off-targeting results in induction of a p53-dependent cell death pathway. p53 activation has also been shown to be an unspecified off-target effect of siRNAs. Both commonly used knockdown technologies can thus induce secondary but sequence-specific p53 activation. p53 inhibition could potentially be applicable to other systems to suppress off-target effects caused by other knockdown technologies. The study shows that p53 MO can be used to attenuate off-target effects of MOs and facilitate the study of specific loss of function phenotypes. p53 MO also helps in studying craniofacial development by reducing off-target effects. The study also shows that p53 activation is induced by MOs through the induction of an N-terminal truncated p53 isoform. The results suggest that p53 activation is a common feature of off-target effects caused by various knockdown technologies. The study provides both a new gene knockdown enhancement tool as well as additional insight into an important and conserved pathway implicated in cellular toxicity.Morpholino phosphorodiamidate antisense oligonucleotides (MOs) and short interfering RNAs (siRNAs) are commonly used to study gene function through sequence-specific knockdown. However, both technologies can cause off-target effects. This study shows that MOs induce off-target effects through p53 activation, leading to cell death. The major off-target effect is mediated by p53 activation, as detected by TUNEL assay, acridine orange, and p21 transcriptional activation. Concurrent knockdown of p53 ameliorates the cell death caused by MO off-targeting. Interestingly, p53 knockdown does not affect specific gene function loss, such as the cell death caused by loss of function of chordin. MO off-target effects are accompanied by diagnostic transcription of an N-terminal truncated p53 isoform. This study shows that MO off-targeting results in induction of a p53-dependent cell death pathway. p53 activation has also been shown to be an unspecified off-target effect of siRNAs. Both commonly used knockdown technologies can thus induce secondary but sequence-specific p53 activation. p53 inhibition could potentially be applicable to other systems to suppress off-target effects caused by other knockdown technologies. The study shows that p53 MO can be used to attenuate off-target effects of MOs and facilitate the study of specific loss of function phenotypes. p53 MO also helps in studying craniofacial development by reducing off-target effects. The study also shows that p53 activation is induced by MOs through the induction of an N-terminal truncated p53 isoform. The results suggest that p53 activation is a common feature of off-target effects caused by various knockdown technologies. The study provides both a new gene knockdown enhancement tool as well as additional insight into an important and conserved pathway implicated in cellular toxicity.