Abstract A randomized complete block design was used to evaluate in vitro total gas production (TGP), methane production, concentration of NH3-N, and digestibility of mixed winter forage (CP 10.2% and NDF 58.6%) incubated with calcium-ammonium nitrate (CAN). In vitro fermentation consisted of 50 mL of a 4:1 buffer:ruminal fluid inoculum and 0.7 g of substrate [DM; wheat, triticale, and rye (Triticum aestivum, Triticosecale rimpaui, and Secale cereal; FOR) or forage:corn (90:10; CORN)] incubated for 48 h. Treatments included: 1) FOR; 2) CORN; 3) CORN + 2% CAN (DM; NIT); and 4) CORN + 0.67% UREA (DM; UREA). Treatments NIT and UREA were isonitrogenous. In vitro organic matter digestibility (IVOMD) was determined after incubation for 48 h, followed by a 48-h incubation with HCl and pepsin solutions. Data were analyzed using the MIXED procedure of SAS with the fixed effect of treatment and random effect of day (block). There was a treatment effect (P = 0.024) on TGP where NIT decreased TGP compared with CORN (P = 0.023), and NIT was not different from UREA. A greater IVOMD (P = 0.017) was observed for CORN compared with FOR. No differences were observed in IVOMD between UREA and NIT. There were no differences (P = 0.727) among all treatments for concentration of NH3-N. Total methane production was lesser for NIT (P ≤ 0.018) compared with all other treatments. There was a treatment effect for molar proportion of acetate (P = 0.039) and acetate:propionate (P = 0.034) where NIT tended (P = 0.058) to have a greater molar proportion of acetate compared to UREA. Total VFA concentration was not affected by treatment (P = 0.454). Calcium-ammonium nitrate influenced in vitro ruminal fermentation of a mature mixed winter forage, decreasing methane production without negatively affecting IVOMD or concentration of VFA.