Trimethoprim (TMP) is an antibacterial agent, a lipophilic weak base, and its chemical structure belongs to the pyrimethamine class. It has antibacterial activity against Escherichia coli, Klebsiella, Proteus mirabilis, Salmonella, and Shigella, but has no obvious antibacterial effect on Streptococcus pneumoniae, Neisseria gonorrhoeae, and Neisseria meningitidis, and has no effect on Pseudomonas aeruginosa. The mechanism of action of this product is to interfere with bacterial folic acid metabolism. It mainly selectively inhibits the activity of bacterial dihydrofolate reductase, so that dihydrofolate cannot be reduced to tetrahydrofolate, and synthetic folic acid is the main component of nucleic acid biosynthesis. Therefore, this product prevents the synthesis of bacterial nucleic acids and proteins, and the binding of this product to bacterial dihydrofolate reductase is 50,000 to 60,000 times tighter than that to mammalian enzymes. The combination of this product and sulfonamides can double block the bacterial folic acid synthesis metabolism, have a synergistic effect, enhance the antibacterial activity of sulfonamides, and convert the antibacterial effect into a bactericidal effect, reducing the production of drug-resistant strains.

It has high antibacterial activity against common aerobic Gram-positive bacteria, such as Staphylococcus aureus (including those resistant to penicillin G), Staphylococcus epidermidis, beta-hemolytic Streptococcus, viridans Streptococcus and Streptococcus pneumoniae. It has good antibacterial effect on anaerobic bacteria, including Clostridium diphtheriae, and Clostridium perfringens. It has no activity against Gram-negative bacteria such as enterococci, meningococci, Neisseria gonorrhoeae, and Haemophilus influenzae, as well as fungi. There is no cross-resistance with penicillin, chloramphenicol, cephalosporins and tetracyclines, and there is partial cross-resistance with macrolides. It acts on the 50S subunit of the ribosome of sensitive bacteria, preventing the extension of the peptide chain, thereby inhibiting the protein synthesis of bacterial cells. It is generally an antibacterial agent, but at high concentrations, it also has a bactericidal effect on certain bacteria.

It combines with blood ammonia in the body to form urea and is excreted from the body, which has the effect of reducing blood ammonia and promoting brain metabolism. It may be a central mediator that can enhance the activity of glucose phosphatase and facilitate the recovery of brain cell function.

It combines with adenosine triphosphate to form vitamin B1 pyrophosphate (thiamine diphosphate, cocarboxylase), which is a coenzyme necessary for carbohydrate metabolism; it can inhibit the activity of cholinesterase. When it is deficient, the activity of cholinesterase is enhanced, the hydrolysis of acetylcholine is accelerated, causing nerve impulse conduction disorders, and affecting gastrointestinal and myocardial function.