Arsenic (+3 oxidation state) methyltransferase (AS3MT) catalyzes the S-adenosylmethionine (SAM)-dependent methylation of inorganic arsenic (iAs), yielding monomethyl‑arsenic (MAs) and dimethyl‑arsenic (DMAs) metabolites. The formation of DMAs in this pathway is considered a key mechanism for iAs detoxification. Availability of SAM for iAs methylation depends in part on dietary intake of folate. Results of population studies suggest that supplementation with folate stimulates iAs methylation, increasing DMAs and decreasing iAs and MAs proportions in urine and/or blood. The goal of the present study was to determine if folate intake affects methylation and clearance of iAs in a recently established mouse strain that expresses human AS3MT and exhibits a human-like pattern of iAs metabolism. The humanized male and female mice were fed folate-deficient (FD) or folate-supplemented (FS) diet for 6 weeks, followed by exposure to 0 ppb or 400 ppb iAs in drinking water for 5 weeks, while on the same types of diet. The concentrations and proportions of iAs, MAs and DMAs were determined in urine, liver, kidneys, and spleen. The diet-, sex- and dose-related differences were assessed by t-test or a non-parametric test; Bonferroni test was used to correct for multiple comparisons. In general, proportions of DMAs were greater and proportions of iAs were smaller in urine and tissues of FS mice as compared to FD mice. However, folate supplementation also increased MAs proportions. Notably, the folate intake had no effect on the concentrations of total arsenic either in the urine or the tissues. These results suggest that, similar to humans, folate supplementation stimulates iAs methylation in the humanized mice. However, the stimulation of iAs methylation is not associated with clearance of arsenic from tissues, possibly due to an inefficient conversion of MAs to DMAs.