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Abstract Background Spinal cord atrophy occurs early in multiple sclerosis (MS) and impacts disability. The therapeutic effect of interferon beta-1a (IFNβ-1a) on spinal cord atrophy in patients with relapsing-remitting (RR) MS has not been explored. Methods We retrospectively identified 16 consecutive patients receiving weekly intramuscular IFNβ-1a for 2 years [baseline age (mean ± SD) 47.7 ± 7.5 years, Expanded Disability Status Scale score median (range) 1.5 (0–2.5), timed 25-foot walk 4.6 ± 0.7 seconds; time on treatment 68.3 ± 59.9 months] and 11 sex- and age-matched normal controls (NC). The spinal cord was imaged at baseline, 1 and 2 years later with 3T MRI. C1-C5 spinal cord volume was measured by an active surface method, from which normalized spinal cord area (SCA) was calculated. Results SCA showed no change in the MS or NC group over 2 years [mean annualized difference (95 % CI) MS: −0.604 mm 2 (−1.352, 0.144), p = 0.106; NC: −0.360 mm 2 (−1.576, 0.855), p = 0.524]. Between group analysis indicated no differences in on-study SCA change [MS vs. NC; year 1 vs. baseline, mean annualized difference (95 % CI) 0.400 mm 2 (−3.350, 2.549), p = 0.780; year 2 vs. year 1: −1.196 mm 2 (−0.875, 3.266), p = 0.245; year 2 vs. baseline −0.243 mm 2 (−1.120, 1.607), p = 0.712]. Conclusion Established IFNβ-1a therapy was not associated with ongoing spinal cord atrophy or any difference in the rate of spinal cord volume change in RRMS compared to NC over 2 years. These results may reflect a treatment effect. However, due to sample size and study design, these results should be considered preliminary and await confirmation.