To investigate the physiological function of thiamine I in myxobacteria, Corallococcus sp. EGB, Myxococcus xanthus DK1622 and Cystobacter sp. 1404 were selected and employed as research subjects in this study. Genes related to thiamine synthesis pathway in the genomes of three stains and their relationships with the growth and development of strains were studied. The results showed that all three strains’ genomes contained complete thiamine synthesis pathway and genes related to thiamine synthesis precursor pyrimidine (HMP) recovery, but did not contain genes related to thiamine or its precursor thiazole recovery. The TPP-riboswitch present upstream of the HMP synthase gene thiC can regulate the transcriptional level of the thiC gene according to the thiamine concentration. Mutant CL1006 and CL1007 were constructed by inserting the thiC gene into strain DK1622 and thiaminase I knockout mutant CL1003, respectively. CL1006 required additional thiamine or HMP for growth in thiamine-free medium, with a significant increase of 9.0% in colony diameter compared to the thiamine-treated group when HMP was added alone. CL1007 could only grow on medium supplemented with HMP, and the addition of intact thiamine alone could not restore its growth. However, when both CcThi1 and thiamine were added, the growth of CL1007 was restored. These results indicate that myxobacteria do not directly utilize exogenous thiamine but could utilize pyrimidine precursors produced by decomposing thiamine through thiaminase I. This finding lays a foundation for elucidating the physiological function of thiamine I in myxobacteria and in-depth analysis of the ecological regulation mechanism of myxobacteria on microbial communities.