The present study investigated the effects of long-chain fatty acids with different degrees of unsaturation on rumen protozoal engulfment and microbial protein recycling by protozoa in vitro. The seven experimental treatments included stearic acid (C18:0, Group A), oleic acid (C18:1, n-9, Group B), linoleic acid (C18:2, n-6, Group C), α-linoleic acid (C18:3, n-3, Group D), arachidonic acid (C20:4, n-6, Group E), eicosapentaenoic acid (C20:5, n-3, Group F) and calcium palmitate (C16:0, control group, G), each being included at 3% (w/w) in the total culture substrate containing starch, xylan, araban, glucan, mannan, cellulose, pectin, lignin, urea and casein. Three goats fitted with rumen cannula were used to provide rumen fluid. The incubation medium was collected for the measurement of engulfing rate of bacteria by protozoa and microbial biomass after 24 h of in vitro incubation. The results showed that the bacterial density of Group D (5.75 × 109 cells/mL) was significantly higher than that of Groups A, B, E, F and G (P < 0.05), but that of the control (Group G) as well as those of Groups A and B were lower than those of Groups D and C (P < 0.05). Similarly, the bacterial protein was the highest in Group D and the lowest in Group G. The number of bacteria engulfed by protozoa per millilitre were the highest in the Control group G (847 × 105 cells/(mL.h)) and the lowest in Group D (392 × 105 cells/(mL.h)). The recycling rate of bacterial cells was lowest in Group D (0.68%) and the recycling time of bacterial cells was the longest (147 h). The quantity of protein recycled was lowest in Group D and highest in Group G, which derived from the number of bacterial cells engulfed. Therefore, it was concluded that the effects of long-chain fatty acids on rumen microbial protein recycling and microbial protein synthesis mainly relate to their degree of unsaturation, with α-linoleic acid possessing a better ability to suppress bacterial-cell (by protozoa) and reduce protein yield.