Human milk has anti-inflammatory characteristics. In a rat model of acetic acid-induced colitis, human milk feeds before and after injury decrease the acute inflammatory response in the colon. As an in vitro correlate of these animal studies, we have developed a rat intestinal epithelial cell (RIE-1) model in which we induce cellular injury with acetic acid and examine human milk- mediated protection. RIE-1cells, a non-transformed small intestinal cell line, were grown in DMEM high glucose media with 10% FBS in 96-well plates for 6 hours, then fed media ±25% aqueous milk (v/v) and allowed to grow in log phase. At 24 hours the cells were briefly exposed (10 sec) to varying concentrations of acetic acid (0.8%-1.2%), returned to media ±25% aqueous milk and allowed to continue growth for 24 hours. Four experimental conditions were examined: cells without any milk exposure (NO MILK); cells exposed to milk before acid injury (MILK/NO MILK); cells exposed to milk after acid injury (NO MILK/MILK), and cells exposed to milk at all times(MILK/MILK). Cell densities in triplicates of each experimental condition were assessed with 0.1% crystal violet staining, solubilization in 0.05M NaH2PO4 in 50% ethanol, and color intensity read at 550λ. Results are expressed as the percent cell density compared to cells under the same experimental conditions without an acid injury. Overall, cells exposed to milk in any way without acetic acid injury were not as dense as cells remaining in DMEM/FBS: NO MILK 0.56±0.2 OD units, n=8; NO MILK/MILK 0.42±0.02, n=8; MILK/NO MILK 0.43±0.03, n=5, and MILK/MILK 0.39±0.03, n=4. A dose response to acetic acid challenge (0.8%-1.2%) was seen in all experimental conditions with cell density falling to 14 -28% of no injury cell density levels at acetic acid doses of 1%-1.2%. However, at the lower acetic acid challenge doses (<1.0%), cell densities in the MILK/NO MILK condition were significantly higher (62±4%, n=10, p=0.015 vs NO MILK) than in the NO MILK (49±3%, n=16), NO MILK/MILK(52±4%, n=13) or MILK/MILK (47±3%, n=8) conditions. These data suggest that 1) milk exposure appears to function “protectively” rather than therapeutically against acetic acid injury; 2) the RIE-1 cell model may have applicability for studying milk protection against chemical injury; and 3) the observed effects may contibute to the anti-inflammatory character of milk.