Folate is an essential dietary vitamin involved in methylation reactions and DNA synthesis. Insufficient dietary folate has been associated with various developmental and adult diseases such as neural tube defects and colorectal cancer. The basis for these associations has not been clearly defined. In microarray studies, components of the Wnt signaling pathway have been shown to be altered under folate-deficient conditions. The Wnt signaling pathway has multiple functions, affecting cell growth, differentiation, and development. The purpose of this study was to determine if folate deficiency leads to altered Wnt signaling with a focus on β-catenin activity, protein levels, and cellular localization. NIH3T3 cells containing the TOPFlash β-catenin luciferase reporter were grown for 4, 8, and 10 days in folate-deficient and sufficient custom DMEM medium either with or without activation of the Wnt pathway by addition of Wnt3a conditioned medium. β-catenin activity, as measured by luciferase activity per unit DNA, was higher in folate-deficient cells at 8 and 10 days in Wnt3a stimulated cells. β-catenin protein levels as assayed by Western blot analysis were not significantly different in folate-deficient cells. Indirect immunofluorescence revealed increased nuclear localization of β-catenin in folate-deficient cells of both Wnt3a stimulated and unstimulated cells at 10 days. Overall, these results suggest that folate deficiency alters cell function, in part, due to changes in Wnt signaling.