In this experiment in vitro and in vivo trials were performed to investigate the efficacy of whole cottonseed to limit protozoal population and fermentation parameters. Treatments were I) control (without whole cottonseed) and 2) 20% whole cottonseed (WCS). Dry matter disappearance (DMD) and fermentation characteristics of the treatments were determined in the in vitro incubation studies. In the in vivo trial, all sheep were fed a control diet for 3 wk, prior to the start of the 28-day experimental period, in a completely randomize design. Ruminal fluid was taken by rumenocentesis (3 h after feeding) on days 1,3,5, 7, 9, 11, 14, 21 and 28 from four sheep fed treatment diets. The pH and protozoal counts were determined on each ruminal fluid sample was taken from sheep, while ammonia nitrogen and volatile fatty acids (VFA) were determined in samples taken on days 7, 14, 21 and 28. The in vitro DMD at 72 h incubation was higher (P<0.01) for the control diet. After 72 h of incubation, the total VFA concentration (mM) (P<0.05) and the acetate to propionate ratio (P<0.05) was higher for the control diet than WCS diet. After 24 h of incubation, the molar proportion (%) of acetate, propionate and valerate (P<0.05) were, higher, but the molar proportion of isovalerate was lower (P<0.05) for the control diet than for the WCS diet. In in vivo study, feeding cottonseed decreased (P<0.05) total protozoa population from approximately 450,000 to 240,000 ml-1 Holotrich and cellulolytic protozoa disappeared from the rumen samples of sheep whereas Entodinium sp., remained. This was associated with lower concentrations of ammonia nitrogen in rumen fluid of sheep fed WCS diet (P<0.01). In vivo molar proportion of propionate was higher and the acetate to propionate ratio was lower for control diet than for WCS diet. It was concluded that feeding whole cottonseed reduced rumen fauna, ammonia nitrogen and the molar proportion of propionate and increased the acetate to propionate ratio, but had no effect on ruminal pH and total VFA concentration.