Cancer cells utilize vitamin folate to fulfill their excessive demand for nucleotides and amino acids. Dihydrofolate reductase (DHFR), an enzyme involved in folate metabolism converts dihydrofolate into tetrahydrofolate, which is required for the de novo synthesis of purines, and certain amino acids. DHFR inhibitors are used as a chemotherapeutic agent. Cancer sequencing analysis has identified additional enzymes in folate metabolism that are dysregulated in cancer. Methylenetetrahydrofolate dehydrogenase 1 like (MTHFD1L), one such enzyme is overexpressed in bladder cancer. MTHFD1L is a mitochondrial enzyme involved in the folate cycle by catalyzing the reaction of formyl-tetrahydrofolate to formate and tetrahydrofolate (THF). THF is crucial for de novo purine and thymidylate synthesis and is also involved in the regeneration of methionine. Cancer cells rely on purines derived from the de novo pathway for the nucleotides whereas normal cells favor the salvage pathway. In this study we examined MTHFD1L expression in bladder cancer. By using publicly available cancer transcriptome data analysis web-portal UALCAN, we found overexpression of MTHFD1L in bladder cancer and expression was associated with overall survival. RT-PCR and immunoblot analysis confirmed the overexpression of MTHFD1L in muscle invasive bladder cancer tissues compared to normal urothelium. Furthermore, our investigations suggested a critical role for MTHFD1L in bladder cancer cell proliferation, colony formation and invasion. Thus, in this study, we show the significance of the folate metabolic enzyme MTHFD1L in aggressive bladder cancers and suggest that being an enzyme, MTHFD1L serves as a valuable therapeutic target.