Background: Rheumatoid arthritis (RA) is an inflammatory disease requiring treatment by disease-modifying agents like methotrexate. C3435T is a single nucleotide polymorphism in the ABCB1 gene that affects the expression of the P-glycoprotein responsible for the efflux of several drugs from cells increasing both its action and toxicity. Objective: Examine the frequency of C3435T in RA patients and assess its influence on responsiveness to methotrexate treatment and occurrence of methotrexate adverse effects. Patients and Methods: Genotyping C3435T polymorphism was done by real-time polymerase chain reaction and the frequency of the polymorphism was assessed in a sample of 90 RA patients who have received methotrexate treatment and 90 healthy controls. The prevalence of the polymorphism was also assessed in responders to methotrexate compared to non-responders, and also in patients who suffered from methotrexate side effects compared to those who did not. Results: The CC was the most frequent genotype in patients (47.8%) while the CT was the most frequent in controls (43.3%), however, no association was found between the ABCB1 C3435T genotype and susceptibility to RA under the different genetic models (p>0.05). Even though the patients who responded to methotrexate had a higher frequency of the T allele (38.9%) compared to non-responders (27.8%), the genotype and allele frequency were not associated with response to methotrexate (p>0.05). Also, no association was observed between the frequency of the C3435T polymorphism and experiencing methotrexate-associated adverse effects (p>0.05).  Conclusion: The C3435T polymorphism of the ABCB1 gene may not be a genetic risk factor for RA and does not affect responsiveness to methotrexate or affect the occurrence of adverse effects from the drug.