Although cattle can synthesize vitamin C (VC) endogenously, stress may increase VC requirements above the biosynthetic threshold and warrant supplementation. This study investigated the effects of a VC injection delivered before or after a long-distance transit event on blood parameters and feedlot performance of beef steers. Fifty-two days prior to trial initiation, 90 newly weaned, Angus-based steers from a single source were transported to Ames, IA, USA. On day 0, 72 steers (356 ± 17 kg) were blocked by BW and randomly assigned to intramuscular injection treatments (24 steers/treatment): saline injection pre- and post-transit (CON), VC (Vet One, Boise, ID, USA; 5 g sodium ascorbate/steer) injection pre-transit and saline injection post-transit (PRE) or saline injection pre-transit and VC injection post-transit (POST). Following pre-transit treatment injections, steers were transported on a commercial livestock trailer for approximately 18 h (1675 km). Post-transit (day 1), steers were sorted into pens with one GrowSafe bunk/pen (4 pens/treatment; 6 steers/pen). Steers were weighed on day 0, 1, 7, 30, 31, 56 and 57. Blood was collected from 3 steers/pen on day 0, 1, 2 and 7; liver biopsies were performed on the same 3 steers/pen on day 2. Data were analyzed as a randomized complete block design (experimental unit = steer; fixed effects = treatment and block) and blood parameters were analyzed as repeated measures. A pre-transit VC injection improved steer average daily gain from day 7 to 31 (P = 0.05) and overall (day 1 to 57; P = 0.02), resulting in greater BW for PRE-steers on day 30/31 (P = 0.03) and a tendency for greater final BW (day 56/57; P = 0.07). Steers that received VC pre- or post-transit had greater DM intake from day 31 to 57 (P = 0.01) and overall (P = 0.02) v. CON-steers. Plasma ascorbate concentrations were greatest for PRE-steers on day 1 and POST-steers on day 2 (treatment × day; P < 0.01). No interaction or treatment effects were observed for other blood parameters (P ≥ 0.21). Plasma ferric-reducing antioxidant potential and malondialdehyde concentrations decreased post-transit (day; P < 0.01), while serum non-esterified fatty acids and haptoglobin concentrations increased post-transit (day; P < 0.01). In general, blood parameters returned to pre-transit values by day 7. Pre-transit administration of injectable VC to beef steers mitigated the decline in plasma ascorbate concentrations and resulted in superior feedlot performance compared to post-transit administration.