In the present study, we determined the effects of long-term exposure to aspirin on the growth and functionality of Lactobacillus rhamnosus. One isolated colony of L. rhamnosus was propagated three times in deMan, Rogosa and Sharpe (MRS) broth and incubated at 37°C. The active strain was then harvested and washed with 0.1% peptone water. Cells were transferred into 9 mL MRS broth containing approximately 6 mg/mL of aspirin, vigorously mixed and incubated for 4 h. The cells were further harvested, transferred into MRS broth and incubated at 37°C. The exposure protocol was repeated for five sequential transfers a week, and the exposed strain cell was surface plated onto MRS agar containing equal amount of aspirin. One isolated colony of aspirin exposed L. rhamnosus from an aspirin containing agar plate was further activated in MRS broth. This procedure was repeated sequentially for 12 consecutive weeks. Bacterial populations, beta-galactosidase activity (beta-gal), and protein expression were determined. Bacterial cells unexposed to aspirin were used as the control. Our results showed that L. rhamnosus could survive after long term exposure to a sub-inhibitory concentration of aspirin. The average beta-gal activity of L. rhamnosus in unexposed cells was 153 ± 2.5 Gal U.; however, beta-gal activity was completely inhibited in exposed cells throughout the exposure period. There was approximately 54% more protein expressed in the long term aspirin-exposed strain compared to the short term exposed (4 h) strain as determined by BCA assay. In addition, the SDS-page gel indicated the synthesis of more protein in the exposed L. rhamnosus. The onset of common or chronic illnesses, especially among the elderly population, is often treated with common medications. However, regular the intake of medical drugs such as aspirin could affect beneficial gut microflora. Therefore, foods containing probiotics such as yogurt and other functional foods are important for the maintenance of optimal gastrointestinal health.