Background: Plasmodium vivax serine hydroxymethyltransferase (PvSHMT) catalyzes formation of glycine from L-serine and tetrahydrofolate. Results: Results indicate that PvSHMT can bind to either substrate first. The rate constant of glycine formation is similar to k cat . Conclusion: PvSHMT reaction occurs via a random-order mechanism and glycine formation is the rate-limiting step. Significance: The data are useful for future investigation on inhibition of SHMT for antimalarial drug development.