Introduction: Mutations in the uncharacterised human FAM111B gene are associated with POIKTMP, a rare multi-organ fibrosing disease. Recent studies also reported the overexpression of FAM111B in specific cancers. Moreover, FAM111B mutation screening may prove expensive in under-resourced facilities. Therefore, this study investigated its cellular function and dysfunction and described an inexpensive mutation screening method. Materials and Methods: FAM111B expression was assessed in silico and validated in vitro in cell lines and primary skin fibroblasts from a South African POIKTMP-patient with the heterozygous FAM111B gene mutation: NM_198947.4: c.1861T>G (p. Tyr621Asp or Y621D) by qPCR and western blot. The cellular function of FAM111B was studied in HT1080 using various cell-based functional assays, and the Y621D mutation was genotyped by PCR-RFLP. Results: Expression studies showed upregulated FAM111B mRNA and protein in the cancer cells. High FAM111B expression with robust nuclear localization occurred in HT1080. Additionally, expression data and cell-based assays indicated that FAM111B led to the upregulation of cell migration, decreased cell apoptosis, and modulatory effects on cell proliferation. Y621D mutation showed similar effects on cell migration but minimal impact on cell apoptosis. FAM111B mRNA and protein expression were markedly downregulated (p ≤ 0.05) in the POIKTMP-patient's fibroblasts. The PCR-RFLP method successfully genotyped Y621D gene mutation. Discussion: FAM111B is a cancer-associated nuclear protein: Its modulation by mutations or overexpression may contribute to the malignancy of cancers and POIKTMP/fibrosis and poor clinical outcomes and represents a viable prognostic marker or therapeutic target. Furthermore, the PCR-RFLP method could prove a valuable tool for FAM111B mutation validation or screening in resource-constrained laboratories.