Toll-like receptors (TLRs) are a class of pattern recognition receptors that sense highly conserved pathogen associated antigenic determinants, triggering an innate immune response and subsequently instructing the adaptive immune system so that together, the pathogen can be eliminated. TLRs are widely distributed in human ocular tissues and cell types, and are active players in ocular inflammation. To date, the presence and function of TLRs on human choroidal melanocytes (HCMs), the most abundant choroidal cell type, have not been characterized. The current study investigated the in vitro and in situ expression and functional status of TLRs on HCMs. HCMs were isolated and cultured from post-mortem human donor eyes, and displayed characteristic melanocyte morphology and MART1 expression – a key melanocyte lineage marker up to passage 5 (P5). In vitro experiments used P1 to P4 HCMs from different donor eyes. Initial quantitative real-time PCR (qPCR) analysis revealed that HCMs (n = 3 donors) expressed specific mRNA transcripts for TLR1-10 and MYD88 (a key adaptor protein initiating the TLR signalling pathway). HCMs were stimulated with a set of synthetic TLR specific agonists and the secretion of pro-inflammatory cytokines, MCP-1 and IL-8, at 24 h measured by ELISA (n = 3 donors). The agonists Pam3CSK4 (TLR1/2), Poly I:C (TLR3), LPS (TLR4), Flagellin (TLR5), and FLS-1 (TLR2) induced a significant increase in the production of MCP-1 and IL-8, compared to untreated cells. Application of biotinylated Pam3CSK4 provided in vitro visualization of receptor-agonist interactions for TLR1/2. We confirmed that cultured HCMs (n = 3 donors) expressed TLR1-6 protein using immunocytochemistry and confocal microscopy. The expression and distribution of TLR 1–6 was also studied in human choroid and retinal pigment epithelium (RPE) sections (n = 3 eyes) using immunofluorescence and confocal microscopy. Strong TLR1-6 immunolabelling that co-localized with melanocyte-dense areas (and RPE) was consistently observed; intraluminal and blood vessel-related cells (including endothelial cells) also expressed several TLRs. Taken together these observations show for the first time that HCMs constitutively express a range of functional TLRs, and as such can contribute to choroidal responses during infection and inflammation.