Horizontal gene transfer (HGT) can equip organisms with novel genes， expanding the repertoire of genetic material available for evolutionary innovation and allowing recipient lineages to colonize new environments. However， few studies have characterized the functions of HGT genes experimentally or examined postacquisition functional divergence. Here， we report the use of ancestral sequence reconstruction and heterologous expression in Saccharomyces cerevisiae to examine the evolutionary history of an oomycete transporter gene family that was horizontally acquired from fungi. We demonstrate that the inferred ancestral oomycete HGT transporter proteins and their extant descendants transport dicarboxylic acids which are intermediates of the tricarboxylic acid cycle. The substrate specificity profile of the most ancestral protein has largely been retained throughout the radiation of oomycetes， including in both plant and animal pathogens and in a free-living saprotroph， indicating that the ancestral HGT transporter function has been maintained by selection across a range of different lifestyles. No evidence of neofunctionalization in terms of substrate specificity was detected for different HGT transporter paralogues which have different patterns of temporal expression. However， a striking expansion of substrate range was observed for one plant pathogenic oomycete， with a HGT derived paralogue from Pythium aphanidermatum encoding a protein that enables tricarboxylic acid uptake in addition to dicarboxylic acid uptake. This demonstrates that HGT acquisitions can provide functional additions to the recipient proteome as well as the foundation material for the evolution of expanded protein functions.