The marine roseobacter Phaeobacter sp. strain Y4I synthesizes the blue antimicrobial secondary metabolite indigoidine when grown in a biofilm or on agar plates. Prior studies suggested that indigoidine production may be, in part, regulated by cell-to-cell communication systems. Phaeobacter sp. strain Y4I possesses two luxR and luxI homologous N-acyl-l-homoserine lactone (AHL)-mediated cell-to-cell communication systems, designated pgaRI and phaRI. We show here that Y4I produces two dominant AHLs, the novel monounsaturated N-(3-hydroxydodecenoyl)-l-homoserine lactone (3OHC_12:1-HSL) and the relatively common N-octanoyl-l-homoserine lactone (C₈-HSL), and provide evidence that they are synthesized by PhaI and PgaI, respectively. A Tn5 insertional mutation in either genetic locus results in the abolishment (pgaR::Tn5) or reduction (phaR::Tn5) of pigment production. Motility defects and denser biofilms were also observed in these mutant backgrounds, suggesting an overlap in the functional roles of these systems. Production of the AHLs occurs at distinct points during growth on an agar surface and was determined by isotope dilution high-performance liquid chromatography–tandem mass spectrometry (ID-HPLC-MS/MS) analysis. Within 2 h of surface inoculation, only 3OHC_12:1-HSL was detected in agar extracts. As surface-attached cells became established (at ∼10 h), the concentration of 3OHC_12:1-HSL decreased, and the concentration of C₈-HSL increased rapidly over 14 h. After longer (>24-h) establishment periods, the concentrations of the two AHLs increased to and stabilized at ∼15 nM and ∼600 nM for 3OHC_12:1-HSL and C₈-HSL, respectively. In contrast, the total amount of indigoidine increased steadily from undetectable to 642 μM by 48 h. Gene expression profiles of the AHL and indigoidine synthases (pgaI, phaI, and igiD) were consistent with their metabolite profiles. These data provide evidence that pgaRI and phaRI play overlapping roles in the regulation of indigoidine biosynthesis, and it is postulated that this allows Phaeobacter sp. strain Y4I to coordinate production of indigoidine with different growth-phase-dependent physiologies.