The objective of this study was to evaluate the effects of replacing raw rapeseed cake (RC) with fermented rapeseed cake (FRC) in the diet of dairy cows on methane (CH4) production, ruminal fermentation, and milk production, composition, and fatty acid composition. The Hohenheim gas test (exp. 1) was initially used to evaluate RC and FRC as substrates. Following batch fermentation, an in vitro study (exp. 2) was performed to assess the effects of replacing RC with FRC at 28.75, 57.5, 86.25, and 115 g/kg (FRC25, FRC50, FRC75, and FRC100) in the total mixed rations (TMR). Based on the in vitro results, the control TMR (115 g/kg dry matter (DM) of RC; CONRC) and experimental TMR (115 g/kg DM of FRC; FRC100) were chosen for an in vivo assessment. In exp. 3, four ruminally cannulated cows were used in a replicated 2 (group) × 2 (period) crossover design and fed the TMR ad libitum. In exp. 4, twenty multiparous Polish Holstein-Friesian cows in their mid-lactation (148 ± 26 d in milk) were used in a completely randomized design. The cows were fed a partial mixed ration without the RC and FRC, and the RC and FRC were supplied in a concentrate feeder at 2.65 kg/d/cow. The FRC100 markedly decreased CH4production by 12% and archaeal population without adversely affecting nutrient digestibility. The molar proportion of propionate was increased, and the molar proportion of acetate and butyrate and acetate to propionate ratio were decreased by FRC100. No significant effects on milk production or composition, except an increase in milk urea concentration, were observed in cows fed FRC100. Milk C18:2cis-9,trans-11 concentration was greater, and n-6 to n-3 fatty acid ratio was lower for FRC100 than CONRC.In-situruminal degradation of RC and FRC were explored usingin-saccotechniques (exp. 5). The potential degradation and effective degradability of the DM, organic matter, and crude protein were significantly higher for FRC than RC. These results suggested that FRC could mitigate enteric CH4production by decreasing archaeal abundances without adversely affecting milk production and ruminal fermentation in lactating cows.