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The aim of this work was to study the influence of a diet with probiotic bacteria on the lipid composition of different tissues from Iberian pigs. Cholesterol and fatty acid profiles were measured in liver, subcutaneous fat, and Serratus ventralis muscle samples. Feeding Iberian pigs a mixture of probiotic microorganisms for 45 days prior to slaughter affected the tissue composition. The feeds of the control and probiotic groups were identical except for the added Bacillus licheniformis and Bacillus subtilis (1.28 × 109 cfu/kg feed) in the feed of the probiotic group. The diet with probiotics significantly affected the fatty acid profile of liver, subcutaneous fat and Serratus ventralis muscle samples, but no significant differences were found for the cholesterol content. The livers of the probiotic group contained lower levels of n‐6 fatty acids and higher levels of n‐3 fatty acids. The subcutaneous fat contained lower levels of n‐6 fatty acids. Serratus ventralis muscles from the probiotic group contained a significantly higher percentage of oleic acid than those of the control group.Practical applications: This study deals with the effect of the addition of probiotic bacteria to the diet of pigs on both the cholesterol and fatty acid content. B. subtilis and B. licheniformis can be used in pig feed (Regulation EC no. 2148/2004), but there are no studies about their effects on fresh meat or other tissues, particularly regarding the fat composition. Thus, it is interesting to study how feeding these microorganisms affects the different tissues. Including these microorganisms in the basic diet could serve as a strategy to modify the fat composition and to obtain healthier products from pigs.