The present study investigated the effects of supplementing slow-release urea (SRU; Optigen, Alltech, Nicholasville, KY) in 3 lactation dairy diets with different forage-to-concentrate ratios (F:C) on in vitro ruminal fermentation characteristics. The experiment was performed in a 3 (F:C) × 2 (without vs. with SRU supplementation) factorial design with 4 independent runs of continuous cultures (n = 4). The 3 F:C included high-forage (HF; 64:36), medium-forage (MF; 51:49), and low-forage diet (LF; 34:66) on a DM basis, and alfalfa hay and cone silage were forage sources at equal proportions. Supplementing SRU considerably reduced dietary concentrations of mixture of soybean meal and canola meal (50:50 on a DM basis) in the SRU supplemented diets. Continuous culture apparatus consisted of 700-mL working volume fermentation vessels to measure major fermentation end products. Each culture was offered a diet of 20 g DM/d in 2 equal portions at 0800 and 2000 h. Decreasing forage proportion reduced ruminal pH (P < 0.01), but supplementing SRU did not influence ruminal pH. Manipulating F:C did not affect total VFA concentration; however, under HF supplementing SRU increased total VFA concentration, while SRU supplementation in MF decreased total VFA concentration, leading to an interaction between F:C and SRU (P = 0.02). Decreasing forage proportion decreased acetate proportion, but increased propionate proportion, resulting in a tendency to decrease acetate-to-propionate ratio (P = 0.09). In contrast, SRU supplementation did not affect individual VFA proportions. Decreasing F:C decreased ammonia-N concentration, whereas supplementation of SRU tended to increase concentration of ammonia-N (P = 0.08). While methane production decreased with decreasing F:C (P < 0.01), supplementing SRU tended to increase methane production (P = 0.07) mainly due to its sizable increase in MF, leading to a tendency of F:C × SRU interaction (P = 0.06). Overall results in the current study indicate that supplementing SRU would be more beneficial in HF with enhanced ruminal fermentation evidenced by increased total VFA concentration.