Vitamin D supplementation is associated with numerous health benefits, including boosted immunity, bone health, and overall mental and physical well-being. Studies have also indicated that vitamin D supplementation may reduce the risk of cancer mortality, diabetes, and multiple sclerosis. Vitamin D is also commonly used for low mood (depression) and related mental health issues, but current research results remain inconsistent, and it is uncertain whether supplementation will necessarily improve these problems.

The effectiveness of vitamin D largely depends on the concentration of 25-hydroxyvitamin D (25(OH)D) in the body (an indicator of vitamin D status measured by blood tests). Most of the benefits of vitamin D supplementation only become apparent after the deficiency is corrected.

When the concentration of vitamin D in the blood exceeds 375 nmol/L or 150 ng/mL, it becomes toxic and may cause the following side effects:
- Loss of appetite
- Nausea, vomiting
- Muscle weakness
- Confusion
- Dehydration
Increased thirst
- Kidney stones can cause extremely high levels of vitamin D in the blood, which can even be life-threatening.

Because the skin has a self-limiting mechanism for producing vitamin D, normal sun exposure usually does not raise vitamin D levels to dangerous levels. (However, people exposed to strong sunlight for extended periods, such as lifeguards, may theoretically produce excessively high levels of vitamin D.) However, through long-term intake of high doses of supplements, blood vitamin D levels can easily rise to toxic levels.

While extremely high doses of vitamin D supplementation are generally considered unsafe, the "safe upper limit for moderate supplementation" remains controversial. Some studies indicate that daily intake of approximately 4,000 IU (100 micrograms) of vitamin D for more than six months may increase the following risks:
- Hypercalcemia
- For older adults: Increased risk of falls

Some studies on older adults have found that:
High doses of vitamin D may increase the risk of falls.
- Another study indicated that women who took high doses of vitamin D experienced a decrease in bone density.

Vitamin D works by binding to and activating the vitamin D receptor (VDR). Once vitamin D binds to VDR, VDR acts as a transcription factor, regulating the activity of over 1,000 genes. This "genomic" action is relatively slow, requiring the synthesis of new mRNA and proteins to produce its effect.

Vitamin D can also affect the body through non-genomic pathways, rapidly activating different intracellular signal transduction pathways. Although VDRs have been found on cell membranes, it is still unclear whether the rapid, non-genomic response is triggered by vitamin D receptors on the membrane or by other cellular receptors.

While vitamin D is most commonly known for its role in bone metabolism, vitamin D receptors (VDRs) can actually be found in most cells and tissues throughout the body, meaning it can affect a wide range of physiological functions. The following are additional functions of vitamin D (but not limited to):
- Anti-cancer effects (promotes cancer cell apoptosis or inhibits cancer cell proliferation)
- Brain development
- Cell differentiation
- Anti-inflammatory effect
- Antioxidant effect
- Antibacterial effect (through regulation of immune cells and promotion of antibacterial peptide gene expression)
- Protect blood vessels
- Nervous system health
- Calcium balance regulation