The magnitude, duration and oscillation of cellular signalling pathway responses are often limited by negative feedback loops, defined as an ‘activator-induced inhibitor’ regulatory motif. Within the NF
κ
B signalling pathway, a key negative feedback regulator is I
κ
B
α
. We show here that, contrary to current understanding, NF
κ
B-inducible expression is not sufficient for providing effective negative feedback. We then employ computational simulations of NF
κ
B signalling to identify I
κ
B
α
molecular properties that are critical for proper negative feedback control and test the resulting predictions in biochemical and single-cell live-imaging studies. We identified nuclear import and nuclear export of I
κ
B
α
and the I
κ
B
α
–NF
κ
B complex, as well as the free I
κ
B
α
half-life, as key determinants of post-induction repression of NF
κ
B and the potential for subsequent reactivation. Our work emphasizes that negative feedback is an emergent systems property determined by multiple molecular and biophysical properties in addition to the required ‘activator-induced inhibitor’ relationship.
Odd skipped related 1is a negative feedback regulator of nodal-induced endoderm development
