Background: In the duplication-degeneration-complementation (DDC) model, a duplicated gene has three possible fates: it may lose functionality through the accumulation of mutations (non-functionalization), acquire a new function (neo-functionalization), or each duplicate gene may retain a subset of functions of the ancestral gene (sub-functionalization). The role that promoter evolution plays in retention of duplicated genes in eukaryotic genomes is not well understood. Fatty acid-binding proteins (Fabp) belong to a multigene family that are highly conserved in sequence and function, but differ in their gene regulation, suggesting selective pressure is exerted via regulatory elements in the promoter. A previous report showed that zebrafish fabp1b.1 and fabp1b.2 promoters underwent sub-functionalization of transcriptional control by peroxisome proliferator-activated receptors (PPAR). The fabp1b.1 promoter retained a functional PPAR response element (PPRE), while fabp1b.2 did not. Results: In this study, we describe the divergent PPAR regulation of zebrafish fabp1a, fabp1b.1, and fabp1b.2 promoters from the ancestral fabp1 gene. Promoter evolution was assessed by sequence analysis, and differential PPAR-agonist activation of fabp1 promoter activity in zebrafish liver and intestine ex vivo cells, and in HEK293 cells transiently transfected with wild-type and mutated fabp1promoter-reporter gene constructs. Spotted gar fabp1, representative of the ancestral fabp1, was responsive to both PPARα and PPARγ, and displayed a biphasic response to PPARα activation. Zebrafish fabp1a was PPARα-selective, fabp1b.1 was PPARγ-selective, while fabp1b.2 was not regulated by PPAR. Conclusions: The zebrafish fabp1 promoters underwent two successive rounds of PPAR-selective subfunctionalization leading to retention of three zebrafish fabp1 genes that display stimuli-specific regulation.