Alimentos contendo probióticos são conhecidos como funcionais e têm sido recomendados para indivíduos com hipercolesterolemia. Com o objetivo de avaliar seu efeito na modulação dos níveis de colesterol sérico, foi conduzido um estudo de 28 dias, utilizando-se 90 ratos machos Wistar, distribuídos entre os tratamentos: Padrão, com 30 animais e Controle, LDR (Leite Desnatado Reconstituído) e P (Probiótico), com 20 animais cada. O grupo Padrão recebeu a dieta AIN-93G durante todo o período experimental. Os demais grupos receberam a mesma dieta acrescida de 1% de colesterol e 0,1% de ácido cólico. Do 15º ao 28º dia, o grupo LDR recebeu 0,1mL/dia/animal de leite desnatado reconstituído a 10% de sólidos não gordurosos e o grupo P, recebeu 0,1mL/dia/animal de probiótico contendo 10(10) UFC/mL de Lactobacillus acidophilus NCFM. Avaliou-se o colesterol total, LDL-colesterol e HDL-colesterol e o peso do fígado, imediatamente após o término da administração do probiótico, onde os ratos tiveram o fígado retirado e o sangue coletado por punção cardíaca. A adição de colesterol e ácido cólico à dieta não elevou (p>0,05) os níveis de colesterol do grupo Controle, LDR e P em relação ao grupo Padrão, no entanto, promoveu aumento significativo de peso (p0,05) os níveis de colesterol sérico dos animais, uma vez que estes níveis se mantiveram normais durante todo o período experimental. Observou-se também que a administração de probiótico não impediu o acúmulo de gordura no fígado dos animais que receberam dieta rica em colesterol e ácido cólico.Probiotics are known as functional foods and have been recommended as dietary adjunct for individuals with high serum cholesterol levels. To evaluate probiotics effects on the modulation of serum cholesterol an experiment was carried out during 28 days, using 90 Wistar male rats, distributed in four treatments and individually housed, with initial average weight of 250±32g. The experimental groups were: Standard; Control; LDR and P. The Standard group received the AIN-93G diet during the whole experimental period. The other groups received for 28 days the AIN-93G diet added of 1% of cholesterol and 0,1% of cholic acid. Of the 15th to the 28th day (after an initial period of 14 days of hipercolesterolemia induction) the LDR group received the supplement of 0,1mL/day/animal of reconstituted skimmed milk at 10% of non fat solids and, the group P received 0,1mL/day/animal of a probiotic in the form of a concentrate of cells contends 10(10)UFC/mL of Lactobacillus acidophilus NCFM. Total cholesterol, high density lipoprotein (HDL) and low density lipoprotein (LDL) were evaluated at 28th days of experiment (immediately after the end of probiotic administration). The dietary increment of 1% of crystalline cholesterol and 0,1% of cholic acid did not promote hipercholesterolemia in the animals, evidenced by the non significant difference (p>0,05) of levels of total cholesterol, LDL-cholesterol and HDL-cholesterol among the groups Standard, Control, LDR and P after 14 days receiving hipercholesterolemic diet. However it provoked significant increase of the weight and alteration of normal coloration of liver (of red intense for red yellowish) due to fat accumulation in this organ, in the animals that received rich diet in cholesterol compared to the Standard group. Thus, rich diet in cholesterol and colic acid did not promote hipercholesterolemia in the animals, but it induced fat liver in the same. In consequence, the consumption of L. acidophilus did not alter (p>0,05) the levels of serum cholesterol of the animals, once these levels stayed normal during the whole experimental period. It was also observed that the probiotic administration did not impede the fat accumulation in the liver of the animals that received rich diet cholesterol and colic acid.