Background: Osteoarthritis is a degenerative joint disease commonly affecting the knee and often begins with joint pain or arthralgia. Vitamin D deficiency has been suggested as a contributing factor to musculoskeletal pain and cartilage metabolism disorders. Indoor workers may be at increased risk due to limited sunlight exposure and prolonged sitting duration. Objective: To compare serum vitamin D levels between arthralgia and non-arthralgia groups among indoor workers with suspected knee osteoarthritis in Kupang City. Methods: An observational analytic study with a cross-sectional design was conducted among 46 indoor workers from several government institutions in Kupang City. Participants were assessed through anamnesis, body mass index measurement, and evaluation of knee pain using the Visual Analogue Scale (VAS). Serum 25-hydroxyvitamin D levels were measured using the electrochemiluminescence immunoassay (ECLIA) method. Subjects were classified into arthralgia and non-arthralgia groups. Data were analyzed using the independent t-test. Results: All participants were female, aged 18–59 years. Of the 46 respondents, 22 were classified as having arthralgia and 24 as non-arthralgia. A significant difference in serum 25-hydroxyvitamin D levels was observed between the two groups (p < 0.05). The arthralgia group showed a lower mean serum vitamin D level (17.2 ± 2.94 ng/mL) with a mean VAS score of 5.83, indicating moderate pain. In contrast, the non-arthralgia group had a higher mean serum vitamin D level (24.1 ± 2.51 ng/mL) with a VAS score of 0. Conclusion: There was a significant difference in serum vitamin D levels between arthralgia and non-arthralgia indoor workers with suspected knee osteoarthritis in Kupang City, with lower vitamin D levels observed in the arthralgia group.