The sources of bioavailable vitamin B12 are limited, and most of them are animal-derived. Chlorella vulgaris, a freshwater microalga, is known for immune system boosting, nutraceutical properties and presence of a natural form of vitamin B12. The present study focused on the in vivo evaluation of the Chlorella biomass as a source of bioavailable vitamin B12 to alleviate the vitamin B12 deficiency status of Wistar rats. Experimental animals were evaluated for the vitamin B12 deficiency-related circulatory marker (serum vitamin B12) and functional markers (plasma homocysteine and urinary methylmalonic acid), haematological and histological changes. The results showed that an increase of 2.4-fold in urinary methylmalonic acid (13.01 ± 0.89 µmoles moles of creatinine−1), 2.6-fold in plasma homocysteine (17.18 ± 3.57 µmole L−1), and 48% decrease in serum vitamin B12 levels (252.69 ± 1.46 pg mL−1) in vitamin B12 deficient group compared to control animals. The Chlorella biomass supplementation in the diet led to the restoration of the functional and circulatory markers, hematological parameters, and vitamin B12 content of kidney and liver to control levels. The Chlorella biomass supplementation increased the erythrocyte precursors and MAST cells in the bone marrow and also normalized the histological features of kidney, liver, and lung tissues. The results suggest that the vitamin B12 from the Chlorella biomass was bioavailable and facilitated the improvement of vitamin B12 status in deficient rats. [ABSTRACT FROM AUTHOR]