Dendritic cells (DC) play critical roles in immune-mediated kidney diseases. Little is known, however, about DC subsets in human chronic kidney disease, with previous studies restricted to a limited set of pathologies and to using immunohistochemical methods. In this study, we developed novel protocols for extracting renal DC subsets from diseased human kidneys and identified, enumerated and phenotyped them by multi-colour flow cytometry. We detected significantly greater numbers of total DC, CD141^hi and CD1c⁺ myeloid DC (mDC) subsets in diseased biopsies with interstitial fibrosis than diseased biopsies without fibrosis or healthy tissue. In contrast, plasmacytoid DC numbers were significantly higher in the fibrotic group compared to healthy tissue only. Numbers of all DC subsets correlated with loss of kidney function, recorded as estimated glomerular filtration rate. CD141^hi DC expressed CLEC9A, whilst the majority of CD1c⁺ DC lacked expression of CD1a and DC-SIGN, suggesting these mDC subsets may be circulating CD141^hi and CD1c⁺ blood DC infiltrating kidney tissue. Our analysis revealed CLEC9A⁺ and CD1c⁺ cells were restricted to the tubulointerstitium. Notably, DC expression of the costimulatory and maturation molecule CD86 was significantly increased in both diseased cohorts compared to healthy tissue. TGF-β levels in dissociated tissue supernatants were significantly elevated in diseased biopsies with fibrosis compared to non-fibrotic biopsies, with mDC identified as a major source of this pro-fibrotic cytokine. Collectively, our data indicate that activated mDC subsets, likely recruited into the tubulointerstitium, are positioned to play a role in the development of fibrosis and thus, progression to chronic kidney disease.