Finding a biomarker of long length-of-stay (LOS) would provide a simple solution to target frail older inpatients at such risk. The aim of this study was to determine whether serum 25-hydroxyvitamin D (25OHD) deficiency, defined as serum concentration <25 nmol/L, was associated with long LOS among inpatients admitted to a geriatric acute care unit. Three hundred and six older inpatients (mean age 84.6 ± 6.4 years, 61.8% women) were consecutively included in this longitudinal prospective cohort study. Patients were separated into two groups according to LOS tertiles: highest tertile (≥14 days; n = 120) versus the other two tertiles combined (<14 days; n = 186). Serum 25OHD deficiency (58.3 vs. 38.7%, P = 0.001) and the prevalence of male gender (58.3 vs. 28.5%, P < 0.001) were higher among inpatients with the highest LOS compared with those with lower LOS. Serum 25OHD deficiency [odds ratio (OR) = 2.22, P = 0.001 for unadjusted model; OR = 1.87, P = 0.012 for fully adjusted model) and male gender (OR = 2.87, P < 0.001 for unadjusted model; OR = 2.64, P = 0.001 for full model) were associated with a high LOS. Vitamin D deficiency and male gender were risk factors for a long LOS in this pilot study. Using these risk factors to identify inpatients at risk of long LOS may be helpful in adapting early care plans, and thus improving inpatients' health and shortening LOS.