<p>Vitamin B12 is a vital water-soluble vitamin containing a central cobalt atom within its corrin ring structure. It exists in several derivatives, among which methylcobalamin (MeCbl) and adenosylcobalamin (AdCbl) are the biologically active forms that serve as cofactors in essential enzymatic reactions. Although the neurological and hematological consequences of vitamin B12 deficiency have been extensively studied, its role in immune regulation remains less well understood. Considering that macrophages are key effector cells of innate immunity, this study aimed to investigate the immunostimulatory and immunomodulatory properties of MeCbl and AdCbl on murine macrophages (J774.2 cell line) with a particular focus on cytokine responses and the JNK signaling pathway. Macrophages were cultured under controlled conditions and treated with non-cytotoxic concentrations of MeCbl and AdCbl (1, 5, and 10&#xa0;µg/mL), either alone or in combination with lipopolysaccharide (LPS, 1&#xa0;µg/mL) as a pro-inflammatory stimulus. Cell viability was first confirmed using Trypan Blue exclusion to ensure that the tested concentrations did not impair cellular survival. Supernatants were then analyzed for cytokine production (TNF-α, IL-6, IL-12p40, GM-CSF) using ELISA, and intracellular signaling was investigated through flow cytometric analysis of phosphorylated JNK (p-JNK) levels. The results revealed that under non-inflammatory conditions, neither MeCbl nor AdCbl significantly altered cytokine production, suggesting a lack of direct immunostimulatory activity in resting macrophages. However, in the presence of LPS, both derivatives markedly increased TNF-α levels, consistent with enhanced pro-inflammatory activation. At the same time, both compounds significantly suppressed IL-6 and IL-12p40 production, pointing to their capacity to counterbalance excessive Th1-driven responses. Importantly, AdCbl demonstrated a unique, concentration-dependent reduction in GM-CSF levels, whereas MeCbl induced only minimal and inconsistent changes, indicating that AdCbl exerts a more selective regulatory profile while MeCbl has broader but less concentration-dependent effects. Flow cytometry further confirmed that both derivatives strongly enhanced JNK phosphorylation, implicating this signaling pathway as a mechanistic link between vitamin B12 derivatives and cytokine modulation. Taken together, these findings demonstrate that MeCbl and AdCbl exert distinct yet overlapping immunomodulatory effects in macrophages. By simultaneously promoting TNF-α while limiting IL-6, IL-12p40, and GM-CSF (for AdCbl), these compounds display a complex balance of pro- and anti-inflammatory activities mediated, at least in part, through the JNK pathway. This dual role highlights their potential relevance in fine-tuning immune responses, preventing excessive inflammation, and supporting host defense. The study provides new insights into the immunological functions of vitamin B12 derivatives and suggests that future research should explore their therapeutic applications in immune-related disorders and inflammatory diseases.</p>