In humans, six common non-synonymous exonic single-nucleotide polymorphisms in ALDH1L1 lead to ten enzyme haplotypes. Since ALDH1L1 is a metabolic regulator and a candidate tumor suppressor, we studied how haplotype variants affect metabotypes, proliferation and motility. Using lentiviral delivery to A549 and PC3 cells, we have generated two cell panels, with each cell line in the panel carrying one of ten ALDH1L1 haplotype variants. Untargeted metabolomics revealed strong differences in metabotypes between cells carrying ALDH1L1 variants. Certain ALDH1L1 variants had decreased ability to regulate one-carbon metabolism and downstream pathways, including amino acid and glutathione metabolism, nucleotide biosynthesis, and the TCA cycle. The direct comparison of A549 and PC3 cells using an untargeted metabolomic dataset also showed that the two cell lines have vastly different metabotypes. In agreement with this finding, the analysis of A549 and PC3 ALDH1L1 haplotype panels suggested that the ALDH1L1-linked metabolic regulation is strongly influenced by specific cellular metabotypes and genetic background. ALDH1L1 variants also exhibited reduced inhibition of cellular proliferation and motility compared to the most common haplotype, which supports further investigation of their potential relevance to individual risk of metabolic disorders.