Background: Diabetic foot ulcers (DFUs), significantly burden health and socioeconomic systems, primarily due to peripheral neuropathy and uncontrolled glucose levels. Emerging research suggests a link to micronutrient deficiencies, notably vitamin D. Despite hypotheses that vitamin D deficiency impedes DFU healing and worsens outcomes, data on this association in our setting are limited. This study therefore aimed to determine the prevalence, severity, and factors associated with low serum vitamin D levels among DFU patients in Western Uganda. Methods: In this cross-sectional study, we recruited participants diagnosed with DFUs who exhibited low serum vitamin D levels. The study utilized bedside vitamin D rapid diagnostic tests to assess these levels, alongside evaluating patient characteristics and applying the Wagner classification system to classify DFUs. Data analysis was conducted using Stata version 14.2, with statistical significance defined as p < 0.05. Results: 109 patients with DFUs were assessed, with 83.5% showing low serum vitamin D levels. Among those with low serum vitamin D levels, Wagner grade 3 ulcers predominated (35.1%). The cohort was predominantly male (72.5%), with a mean age of 58 years; the majority had primary education or lacked formal schooling (74.7%). Additionally, 28.6% reported alcohol use, 24.2% reported smoking, and 68.8% had hypertension. In regression analysis, a history of smoking was associated with low serum vitamin D levels among DFU patients (AOR = 3.168, CI: 1.050-9.561, p = 0.041). A history of having a nephropathy was also associated with low vitamin D levels (AOR=3.630, CI: 1.315-10.02, p=0.01), additionally patients who rarely ate fruits and vegetables were also associated with having low serum vitamin D levels (AOR=3.426, CI: 1.105-10.84, p=0.036), and late Wagner grades of DFUs were associated with low serum vitamin D levels (AOR = 0.187, CI: 0.073-0.478, p < 0.001). Conclusion: Patients with DFU, particularly those with advanced Wagner grades and who smoke, are at increased risk of vitamin D insufficiency or deficiency. Also, patients who have a nephropathy and those that rarely have fruits or vegetables in their diet have an increased risk of having vitamin D deficiency or insufficiency. Recommendations: Based on our findings, we recommend regular monitoring of vitamin D levels in DFU patients, particularly smokers, patients with nephropathy and supplementation for those with suboptimal levels