Two hundred and ten Angus × Simmental steers (initial BW 314 ± 11 kg) were separated into heavy and light BW blocks and allotted evenly by BW to 6 treatments (3 heavy and 2 light pens per treatment) to determine the effect of supplemental vitamin D3: 0 IU (no D), 250,000 IU for 165 d (long-term D), or 5 × 10(6) IU for 10 d (short-term D) on plasma and muscle calcium concentrations and gene expression in steers fed either 0 (NZ) or 8.38 mg/kg (ZH) zilpaterol hydrochloride (ZH) daily for 21 d. Placebo or ZH was added to the diet 24 d, and short-term D was added 13 d before slaughter. Treatments were removed from all diets 3 d before slaughter. Plasma total calcium (Ca(2+)) was determined at study initiation, start of ZH and short-term D feedings, and at vitamin D3 and ZH withdrawal. Both plasma total and ionic Ca(2+) were determined when animals were sent to harvest. Longissimus muscle total and ionic Ca(2+) were determined in meat aged 7 and 4 d postmortem, respectively. When ZH was fed, long-term D decreased plasma total Ca(2+) at slaughter (P < 0.04). Short-term D increased (P < 0.01) plasma total and ionic Ca(2+) at slaughter regardless of ZH inclusion in the diet. Long- and short-term D, with or without ZH, did not affect (P > 0.28) LM total Ca(2+); however, both long- and short-term D increased LM ionic Ca(2+) when ZH was not fed (P < 0.01). Long-term D reduced LM ionic Ca(2+) when ZH was fed (P < 0.02). Neither long- nor short-term D affected PPARα or δ gene expression (P = 0.19) whether or not ZH was fed. Expression of MYH1 and 2A (P < 0.05) but not 2X (P = 0.21) was decreased in steers fed ZH. Long-term D had no effect on MYH2A expression (P = 0.21). Short-term D increased MYH2A expression when ZH was not fed (P < 0.03). Calpain mRNA tended to be lower in steers fed ZH (P = 0.09), but was not affected by long- or short-term D regardless of whether or not ZH was fed (P = 0.39). Expression of calpastatin did not differ with vitamin D supplementation (P = 0.35). In conclusion, ZH decreased oxidative myosin expression, and when combined with long-term D, ZH decreased LM ionic Ca(2+). Moreover, vitamin D3 supplementation did not increase calpain mRNA. These results help explain why vitamin D3 does not improve tenderness in steers fed ZH.