Background Early detection of mobility limitations remains an important goal for preventing mobility disability. The purpose of this study was to examine the association between the Short Physical Performance Battery (SPPB) and the loss of ability to walk 400 m, an objectively assessed mobility outcome increasingly used in clinical trials. Methods The study sample consisted of 542 adults from the InCHIANTI study aged 65 and older, who completed the 400 m walk at baseline and had evaluations on the SPPB and 400 m walk at baseline and 3-year follow-up. Multiple logistic regression models were used to determine whether SPPB scores predict the loss of ability to walk 400 m at follow-up among persons able to walk 400 m at baseline. Results The 3-year incidence of failing the 400 m walk was 15.5%. After adjusting for age, sex, education, body mass index, Mini-Mental State Examination, number of medical conditions, and 400 m walk gait speed at baseline, SPPB score was significantly associated with loss of ability to walk 400 m after 3 years. Participants with SPPB scores of 10 or lower at baseline had significantly higher odds of mobility disability at follow-up (odds ratio OR = 3.38, 95% confidence interval CI: 1.32–8.65) compared with those who scored 12, with a graded response across the range of SPPB scores (OR = 26.93, 95% CI: 7.51–96.50; OR = 7.67, 95% CI: 2.26–26.04; OR = 8.28, 95% CI: 3.32–20.67 for SPPB ≤ 7, SPPB 8, and SPPB 9, respectively). Conclusions The SPPB strongly predicts loss of ability to walk 400 m. Thus, using the SPPB to identify older persons at high risk of lower body functional limitations seems a valid means of recognizing individuals who would benefit most from preventive interventions.