Background and Objectives: Methotrexate (MTX) is a worldwide drug used to treat neoplastic and non-neoplastic diseases despite its severe side-effects. The present study aimed to throw more light on the MTX effects on cardiac muscle of adult male albino rats and the possible protective role of L-citrulline. Materials and Methods: Thirty Adult male albino rats were used and equally divided into three Groups: Group I: control, Group II (MTX treated), Group III (MTX and L-citrulline treated). Left ventricular specimens were prepared for H and E, Mallory trichrome staining, immunohistochemical analysis for TLR2 and electron microscope examination. Results: Cardiac muscles of MTX treated rats (Group II) showed histological, ultrastructural and histochemical changes such as dark stained nuclei, vacuolation, pyknotic nuclei, loss of striations and interrupted intercalated disc. Increased collagen fibers were also found. Minimal changes were observed in Group III (MTX and L-citrulline treated). Conclusion: MTX has a deleterious effect on rat cardiac muscle and administration of L-citrulline has a protective effect.