The effects of a monensin premix on milk fatty acid content during grain-induced subacute ruminal acidosis (SARA) in Holstein cows receiving a total mixed ration was investigated. Six multiparous, rumen-fistulated Holstein cows were used in a two-treatment, two-period crossover design with 6-wk periods. Experimental treatments were either a monensin premix or a placebo premix. At the beginning of wk 4, SARA was induced in experimental cows for a 10-d period using a grain challenge model. The administration of a monensin premix elevated milk fat proportion of total short-chain saturated fatty acids (sum of C4 to C15). Milk fat proportions of conjugated linoleic acid isomers were unaffected. Linolenic acid (C18:3n3) proportion in milk fat of monensin-treated cows were lower when compared with placebo-treated cows during the SARA period. Results from this study indicate that dietary supplementation with monensin during SARA had little effect on milk fatty acid content.