Background: 25-hydroxycholecalciferol also known as 25-hydroxyvitamin D plays a vital role in numerous physiological functions within the human body, and its deficiency is associated with conditions such as osteoporosis, cardiovascular diseases, autoimmune disorders, and certain cancers. In Alaska, sunlight exposure is a critical factor that influences vitamin D levels in the population. Methods: This study involved a review of serum 25-hydroxyvitamin D [25(OH)D] levels from January 2014 to June 2018 for Alaska residents, including individuals aged 18 years or older who were permanent residents of the area. The exclusion criteria comprised acute or chronic liver or kidney dysfunction, anemia, cancer history, absorption deficiency, and vitamin D supplementation exceeding 500 IU/day within 90 days before the blood draw. Results: A total of 304 participants, including 107 males (35.2%) and 197 females (64.8%), with a mean age of 50.88, were included. The overall mean serum level of 25(OH)D was 22.7 ± 9.89 ng/mL. Females exhibited a higher mean level (23.40 ± 10.24 ng/mL) than males (21.03 ± 9.05 ng/mL) (P <0.05). Participants older than 60 years had a higher average level of 25(OH)D (26.52 ± 10.28 ng/mL) compared to those younger than 45 years (19.85 ± 9.27 ng/mL) (P < 0.05). Seasonal variations were observed, with levels below 20.0 ng/mL from October to March, below 30.0 ng/mL from April to May (insufficient), and slightly above 30.0 ng/mL (desirable) from June to August (P < 0.0001). No significant intercommunity differences were found. Only 24.3% of the participants had a desirable vitamin D level. The association between the 25(OH)D level and the solar zenith angle was linear and markedly negative (r = −0.924; P < 0.0001). Conclusions: This study demonstrates seasonal variations in 25(OH)D serum levels among the Alaska population. Further exploration of factors such as skin pigmentation, daily vitamin D intake, and body mass index is necessary to comprehensively understand the variation in 25(OH)D serum levels in this region.