The objective of this study was to evaluate effects of isobutyrate supplementation on rumen fermentation, lactation performance and plasma characteristics of dairy cows. Twenty multiparous second filial generation (F2) cows of a cross between Chinese Jinnan Yellow and Holstein cows at 148±4.5 days in milk and 22.3±0.81kg milk production were used in a replicated 4×4 Latin square experiment. The treatments were: control (without isobutyrate), low (LIB), medium (MIB) and high (HIB) isobutyrate supplementation of 20, 40 and 60g per cow per day, respectively. Experimental periods were 30 days with 15d of adaptation and 15d of data collection. Dry matter (DM) intake was not affected by increasing isobutyrate supplementation, but milk yields were highest for the 40g/d isobutyrate supplementation level, where proportion of milk fat, true protein and lactose were minimized. Ruminal pH (6.38–6.24) and ammonia N (13.8–11.1mg/100ml) were linearly (P