Here we investigated whether the sensitivity of microtubules to severing by katanin is regulated by acetylation of the microtubules. During interphase, fibroblasts display long microtubules with discrete regions rich in acetylated tubulin. Overexpression of katanin for short periods of time produced breaks preferentially in these regions. In fibroblasts with experimentally enhanced or diminished microtubule acetylation, the sensitivity of the microtubules to severing by katanin was increased or decreased, respectively. In neurons, microtubules are notably more acetylated in axons than in dendrites. Experimental manipulation of microtubule acetylation in neurons yielded similar results on dendrites as observed on fibroblasts. However, under these experimental conditions, axonal microtubules were not appreciably altered with regard to their sensitivity to katanin. We hypothesized that this may be attributable to the effects of tau on the axonal microtubules, and this was validated by studies in which overexpression of tau caused microtubules in dendrites and fibroblasts to be more resistant to severing by katanin in a manner that was not dependent on the acetylation state of the microtubules. Interestingly, none of these various findings apply to spastin, because the severing of microtubules by spastin does not appear to be strongly influenced by either the acetylation state of the microtubules or tau. We conclude that sensitivity to microtubule severing by katanin is regulated by a balance of factors, including the acetylation state of the microtubules and the binding of tau to the microtubules. In the neuron, this contributes to regional differences in the microtubule arrays of axons and dendrites.