Overexpression of the inducible heat shock protein 70, Hsp72, has broadly cytoprotective effects and improves outcome following stroke. A full understanding of how Hsp72 protects cells against injury is elusive, though several distinct mechanisms are implicated. One mechanism is its anti-inflammatory effects. We study the effects of Hsp72 overexpression on activation of the transcription factor NF-xcexbaB in microglia combining experimentation and mathematical modeling, using TNFxcexb1 to stimulate a microglial cell line stably overexpressing Hsp72. We find that Hsp72 overexpression reduces the amount of NF-xcexbaB DNA binding activity, activity of the upstream kinase IKK, and amount of IxcexbaBxcexb1 inhibitor phosphorylated following TNFxcexb1 application. Simulations evaluating several proposed mechanisms suggest that inhibition of IKK activation is an essential component of its regulatory activities. Unexpectedly we find that Hsp72 overexpression reduces the initial amount of the RelA/p65 NF-xcexbaB subunit in cells, contributing to the attenuated response. Neither mechanism in isolation, however, is sufficient to attenuate the response, providing evidence that Hsp72 relies upon multiple mechanisms to attenuate NF-xcexbaB activation. An additional observation from our study is that the induced expression of IxcexbaBxcexb1 is altered significantly in Hsp72 expressing cells. While the mechanism responsible for this observation is not known, it points to yet another means by which Hsp72 may alter the NF-xcexbaB response. This study illustrates the multi-faceted nature of Hsp72 regulation of NF-xcexbaB activation in microglia and offers further clues to a novel mechanism by which Hsp72 may protect cells against injury. 