Introduction: The Six Spot Step Test has shown good psychometric properties in terms of validity and reliability in people with multiple sclerosis. Yet, the responsiveness and minimal important change are unknown. The objective was to investigate the responsiveness of the Six Spot Step Test against the perceived change of walking limitations and establish estimates for the minimal important change in people with multiple sclerosis. Methods: The Six Spot Step Test was performed before and after four weeks of specialised multidisciplinary inpatient rehabilitation by 142 adults with mild to severe multiple sclerosis. Responsiveness was determined based on anchor- and distribution-based methods, using the Multiple Sclerosis Walking Scale-12 as external criterion. In a supplementary analysis, the Six-Minute Walking Test was used as an external criterion. Results: The correlation between the baseline (r = 0.56, p < 0.01) and discharge (r = 0.55, p < 0.01) Multiple Sclerosis Walking Scale-12 and Six Spot Step Test scores were acceptable. Furthermore, the change scores were weakly associated (r = 0.1, p = 0.27). This trend was similar for the Six-Minute Walking Test when used as anchor. The smallest detectable change was estimated to 1.7 seconds. An improvement in the Six Spot Step Test exceeding 2.1 (95% CI − 0.9 to 5.0) sec and 4.9 (95% CI 1.2–8.6) sec may be considered clinically important on a group level based on the Multiple Sclerosis Walking Scale-12 and the Six-Minute Walking Test, respectively. Conclusion: In a sample of mild to severely disabled people with multiple sclerosis, the Six Spot Step Test showed fair responsiveness against a subjective and objective criterion, indicating a minimal important change between ≥ 2.1 and ≥ 4.9 seconds, respectively. However, a weak association between the change in the Six Spot Step Test and the subjective and objective external criterion calls for cautious interpretation. Hence, the results should be further verified against a valid external criterion.