Background: Irisin is a novel myokine, adipokine and neurokine which increases energy expenditure and improves glucose tolerance. Both myokine irisin and vitamin D (VD) are players in the musculoskeletal system; However, irisin-vitamin D relationship is still unclear. Objective: Investigating the effect of chronic administration of cholecalciferol (vitamin D₃) on serum irisin level in relation to some metabolic parameters in vitamin D deficient rat model. Material and Methods: Thirty six healthy weaned (21 days) male albino rats (80.70±10.1 gram) were used. Rats were divided randomly into two groups: Group I : Control group (n=12) fed on normal balanced diet for 6 weeks then given  1ml of pure canola oil as a vehicle every other day for 2 weeks via  gavage and Group II: vitamin D-deficient (VDD) group (n=24) fed on vitamin D-deficient diet (20% lactose, 2% Ca, and 1.25% P) for 6 weeks, then subdivided randomly into two equal subgroups: Group IIa (VDD vehicle -treated group) which was given 1ml of pure canola oil as a vehicle every other day for 2 weeks via gavage, and Group IIb (VDD D_3- treated group): which was given 1 ml of 75 µg/ml of vitamin D₃ in 1ml of pure canola oil every other day for 2 weeks.  For all groups, BMI, food intake, serum irisin, 25-hydroxy vitamin D 25-OHVD, calcium, phosphorus, parathrmone hormone (PTH), glucose, insulin and calculated homeostasis model assessment of insulin resistance (HOMA-IR) were estimated. Results: VD deficient diet for six weeks induced significantly decrease in serum 25-OHVD together with insignificant changes in PTH, Ca, P levels, BMI and food intake. Regarding serum irisin levels, they were low in VDD rats in comparison to normal rats.  However, after 2 weeks of treatment with VD₃, serum irisin levels were significantly higher in VDD D₃- treated group than in VDD vehicle- treated rats. These elevated levels of irisin were significantly positive correlated to serum 25-OHVD in VDD vehicle- treated and VD deficient D₃- treated groups. Moreover, serum insulin levels and HOMA-IR were significantly higher in VDD rats than that of normal rats and, they were negatively correlated with serum 25-OHVD and irisin in both VDD vehicle- treated and VDD D₃- treated groups. Conclusion: Hypovitaminosis D was significantly associated with reduced irisin levels and elevated insulin resistance. Moreover, irisin levels significantly elevated after restoration of VD. Both serum irisin and 25-OHVD were negatively correlated with insulin resistance. Hypovitaminosis D-induced metabolic deterioration could be resulted from decreased irisin levels.