The immune benefits of vitamin D
3
supplementation beyond calcium and phosphate maintenance are highly clinically debated. Kidney expression of CYP27B1 is the source of endocrine, circulating 1,25(OH)
2
D
3
(active form of vitamin D) that maintains serum calcium and phosphate. 1,25(OH)
2
D
3
may also be made by the CYP27B1 enzyme in nonrenal cells, like immune cells, in a process driven by cellular availability of 25(OH)D
3
and inflammation. Due to the endocrine nature of 1,25(OH)
2
D
3
in circulation, it is difficult to discern between these 2 sources. We recently created a regulatory deletion model of
Cyp27b1
(M1/M21-DIKO) where mice have normal inflammatory-regulated
Cyp27b1
expression in nonrenal tissues (unlike global
Cyp27b1
-KO) but no expression within the kidney. Here, utilizing on-tissue chemical derivatization and matrix assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI), we investigated the distribution of 1,25(OH)
2
D
3
and 25(OH)D
3
in the kidney, liver, spleen, and thymus. MALDI-MSI demonstrated increased 1,25(OH)
2
D
3
in nonrenal tissues such as the spleen after vitamin D
3
supplementation in M1/M21-DIKO mice. Additionally, from this, we found increased
Il4
and decreased
Tnfa
in the spleen after vitamin D
3
supplementation. Taken together, these data demonstrate nonrenal production of 1,25(OH)
2
D
3
in vivo and provide a consequence of vitamin D
3
supplementation and nonrenal 1,25(OH)
2
D
3
production in cytokine changes.