Macrophages are important host defense cells in ruminant paratuberculosis (Johne’s Disease; JD), a chronic enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP). Classical macrophage functions of pathogen trafficking, degradation, and antigen presentation are interrupted in mycobacterial infection. Immunologic stimulation by 25-hydroxyvitamin D 3 (25(OH)D 3) and 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3) enhances bovine macrophage function. The present study aimed to investigate the role of vitamin D 3 on macrophage phenotype and endosomal trafficking of MAP in monocyte-derived macrophages (MDMs) cultured from JD-, JD+ subclinical, and JD+ clinically infected cattle. MDMs were pre-treated 100 ng/ml 25(OH)D 3 or 4 ng/ml 1,25(OH) 2D 3 and incubated 24 hrs with MAP at 10:1 multiplicity of infection (MOI). In vitro MAP infection upregulated pro-inflammatory (M1) CD80 and downregulated resolution/repair (M2) CD163. Vitamin D 3 generally decreased CD80 and increased CD163 expression. Furthermore, early endosomal marker Rab5 was upregulated 140× across all stages of paratuberculosis infection following in vitro MAP infection; however, Rab5 was reduced in MAP-activated MDMs from JD+ subclinical and JD+ clinical cows compared to healthy controls. Rab7 expression decreased in control and clinical cows following MDM infection with MAP. Both forms of vitamin D 3 reduced Rab5 expression in infected MDMs from JD- control cows, while 1,25(OH) 2D 3 decreased Rab7 expression in JD- and JD+ subclinical animals regardless of MAP infection in vitro. Vitamin D 3 promoted phagocytosis in MDMs from JD- and JD+ clinical cows treated with either vitamin D 3 analog. Results from this study show exogenous vitamin D 3 influences macrophage M1/M2 polarization and Rab GTPase expression within MDM culture.