The absorption of glucose, a fundamental energy source for all living cells requires membrane transporters. Currently, approximately 30% of the protein coding genes in humans are unknown. The glucose transporters appear to be attractive candidates for function investigation since many of them are associated with diseases such as cancer and are currently used as drug targets. GLUT14 was reported as a duplicon of GLUT3 and is exclusively expressed in the testis. The substrate mediated by this transporter was unknown and variations in SLC2A14 were associated to non-testicular diseases such as the Alzheimer’s disease. Taken together, the function of GLUT14 needs to be examined in order to determine its substrate and extend prior knowledge on the disease mechanism. MFSD14A was reported as a novel sugar transporter due to sequence similarities with mouse glucose transporters and a conserved sugar binding motif. Since no functional study was conducted on this gene, it is therefore hypothesized that MFSD14A might function as a novel sugar transporter, with glucose being one of the probable substrates. A microarray study on the gills of Carcinus Maenas showed down-regulation of Mfsd14a by exposure to acidified sea water, revealing the possibility of MFSD14A mediating other substrates involved in acid-base balance. We therefore additionally hypothesized that MFSD14A could mediate the transport of ammonia, a critical compound in acid-base homeostasis. These hypotheses and research gaps were addressed using bioinformatic studies utilizing deposited sequences from databases, functional analysis in the Xenopus laevis oocyte system using radiolabeled substrates, and a Mfsd14a-knockdown zebrafish (Danio rerio). Our studies confirmed that GLUT14 mediates the transport of glucose and dehydroascorbic acid but no MFSD14A-mediated glucose uptake was found. However, the uptake and release of radiolabelled methylamine (a proxy of ammonia) was observed. This result was further confirmed in the zebrafish model since Mfsd14a-knockdown larvae showed a reduction in both total and regional ammonia excretion. We therefore concluded that MFSD14A is a novel ammonia transporter. Since we have determined the substrates for two unknown transporters for the first time, our results will allow further explorations into their roles in glucose and ammonia metabolism, as well as the disease associations.