Rumen protozoa, particularly the most predominant Entodinium spp., engulf rumen bacteria and subsequently recycle the microbial protein intraruminally, substantially lowering the nitrogen utilization efficiency in dairy cows. This study aimed to identify plants that can improve nitrogen utilization efficiency by inhibiting rumen protozoa, especially the genus Entodinium. Four different plants were first screened for their protozoa-suppressing activity using in vitro cultures of rumen fluid. Apple of Sodom (Calotropis procera) was found to be the most anti-protozoal, and it was further evaluated using in vitro cultures of fresh rumen fluid collected from 3 lactating cows, which were fed a typical total mixed ration (TMR). Ground leaves of apple of Sodom at one of the 4 levels (0, 0.007, 0.009, and 0.011 g/mL) were tested in 3 replicate in vitro cultures incubated for 24 h at 39°C. Protozoa were enumerated using microscopic identification and counting, while volatile fatty acid (VFA) concentrations and methane production were determined using gas chromatography. Data were analyzed in a complete randomized design using the PROC GLM procedure of SAS 9.4. Orthogonal polynomial contrasts were done to determine linear and quadratic responses of protozoal cells, individual VFA and methane production. The results showed that apple of Sodom at all the tested doses significantly decreased the counts of Entodinium and Isotricha linearly (P < 0.0001) and had a quadratic effect on the counts of Eudiplodinium (P = 0.0083). Dasytricha, Ophryocolex, and Eudiplodinium were not affected (P > 0.05). Total VFA concentration was increased. Except for acetate, the concentrations of individual VFA were significantly affected linearly (P < 0.05), and butyrate and isobutyrate were also affected quadratically (P < 0.05). Acetate:propionate ratio was also significantly affected linearly (P < 0.0001). Methane emission was increased (P < 0.0138). Overall, Calotropis procera leaves can be used as a feed supplement to inhibit Entodinium and increase nitrogen utilization and fermentation in ruminants.