Three goats fitted with rumen fistula were used as donors of rumen fluid. Substrates were incubated in vitro with buffered rumen fluid for 24 h. Different levels of β-carotene (five groups: 10, 50, 100, 200, 500 mg/l) were added as five treatment groups to determine whether it affected rumen fermentation in vitro as compared with blank samples used as controls. NH₃-N concentrations with 50, 100 and 200 mg β-carotene/l group were found significantly lower than that in blank samples (P<0.01). Microbial protein concentrations in all β-carotene-added groups were higher than in blanks, while those found in 200 and 500 mg/l groups were significantly higher (P<0.05 and 0.01, respectively). Total VFAs seemed not to be affected by added β-carotene (P>0.05), although propionate and butyrate concentrations changed. It is concluded that β-carotene can enhance the utilization of NH₃-N by rumen microorganisms and thus promote their growth in vitro.