This research aimed to investigate the effects of replacing soybean meal with high-oil pumpkin seed cake (HOPSC) on ruminal fermentation, lactation performance, milk fatty acid, and ruminal bacterial community in Chinese dairy cows. Six multiparous Chinese Holstein cows at 105.50 ± 5.24 d in milk (mean ± standard deviation) and 36.63 ± 0.74 kg/d of milk yield were randomly allocated, in a 3 × 3 Latin square design, to 3 dietary treatments in which HOPSC replaced soybean meal. Group 1 was the basal diet with no HOPSC (0HOPSC); group 2 was a 50% replacement of soybean meal with HOPSC and dried distillers grains with solubles (DDGS; 50HOPSC), and group 3 was a 100% replacement of soybean meal with HOPSC and DDGS (100HOPSC). We found no difference in the quantity of milk produced or milk composition among the 3 treatment groups. Feed efficiency tended to increase linearly as more HOPSC was consumed. In addition, rumen fermentation was not influenced when soybean meal was replaced with HOPSC and DDGS; the relative abundance of ruminal bacteria at the phylum and genus levels was altered. We also observed that as the level of HOPSC supplementation increased, the relative abundance of Firmicutes and Tenericutes linearly increased, whereas that of Bacteroidetes decreased. However, with increasing HOPSC supplementation, the relative abundance of Ruminococcus decreased linearly at the genus level in the rumen, and the relative abundance of Prevotella showed a linear downward tendency. Changes in dietary composition and rumen bacteria had no significant effect on the fatty acid composition of milk. In conclusion, our results indicated that replacing soybean meal with a combination of HOPSC and DDGS can meet the nutritional needs of high-yielding dairy cows without adversely affecting milk yield and quality; however, the composition of rumen bacteria could be modified. Further study is required to investigate the effects of long-term feeding of HOPSC on rumen fermentation and performance of dairy cows.