Neurovascular coupling studies are widely conducted in anesthetized animals using functional magnetic resonance imaging (fMRI). In this study, the dose-dependent effects of isoflurane on neurovascular coupling were examined with concurrent recordings of the local field potential (FP) and cerebral blood flow (CBF) in the rat somatosensory cortex. Electrical forepaw stimulation was used, and consisted of either a single pulse or 10 pulses at various frequencies. We observed that the FP response to single-pulse stimulation remained unaffected across the different levels of isoflurane tested (1.1–2.1%), whereas the CBF response to single-pulse stimulation increased dose-dependently (7 ± 3% to 17 ± 4%). The isoflurane dose did not affect the vascular reactivity induced by a hypercapnic challenge. These findings suggest that the action of isoflurane affects the neurovascular mechanisms. For 10-pulse stimulation, the summation of the evoked FP responses monotonically decreased with an increase in the isoflurane dose, possibly due to enhancement of the neural adaptation. In contrast, the dose-dependent effect on the CBF response varied with the stimulus frequency; a dose-dependent decrease in the CBF response was observed for high-frequency stimulation, whereas a dose-dependent increase was observed for low-frequency stimulation. Furthermore, a linear time-invariant model consisting of the single-pulse hemodynamic impulse response convoluted with 10-pulse FP recordings showed that the neurovascular transfer function was altered by the isoflurane dose for high-frequency stimulation. These results indicate that careful and consistent maintenance of the depth of anesthesia is required when comparing fMRI data obtained from different animals or physiological and pharmacological manipulations.