Patients with multiple sclerosis (MS) can benefit from treatment with interferon β–1b. However, the mechanisms of action of this drug are incompletely understood and effects of interferon β–1b on axonal injury are not known. A measure of axonal injury can be obtained in vivo using magnetic resonance spectroscopy to quantify the resonance intensity of the neuronal marker, N-acetylaspartate (NAA). In a small pilot study, we performed combined magnetic resonance imaging and magnetic resonance spectroscopic imaging on 10 patients with relapsing-remitting MS before and 1 year after starting treatment with subcutaneous interferon β–1b. Resonance intensities of NAA relative to creatine (Cr) were measured in a large, central brain volume. These measurements were compared with those made in a group of 6 untreated patients selected to have a similar range of scores on the Expanded Disability Status Scale and mean NAA/Cr at baseline. NAA/Cr in the treated group [2.74 (0.16), mean (SD)] showed an increase of 5.5 % 12 months after the start of therapy [2.89 (0.24), p = 0.05], while NAA/Cr in the untreated group decreased, but not significantly [2.76 (0.1) at baseline, 2.65 (0.14) at 12 months, p > 0.1]. NAA/Cr had become significantly higher in the treated group at 12 months than in the untreated group (p = 0.03). Our data suggest that, in addition to losing axons, patients with chronic multiple sclerosis suffer from chronic, sublethal axonal injury that is at least partially reversible with interferon β–1b therapy.