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Twelve ICOs generated from liver resections and biopsies were assessed for metabolic markers and functional HBV entry receptor expression throughout differentiation. Structural changes relevant to HBV infection were characterized using histology, confocal, and electron microscopy examinations. Optimal ICO culture conditions for HBV infection using HepAD38 (genotype D) and plasma derived HBV (genotype B &amp; C) were described. HBV infection was confirmed using HBcAg immunostaining, qRT\u2010PCR (RNA, cccDNA, extracellular DNA), and ELISA (HBsAg and HBeAg). Drug response to antiviral and immunosuppressive agent, and cellular responses (interferon\u2010stimulated genes [ISG]) to interferon\u2010\u03b1 and viral mimic (PolyI:C) were assessed. ICOs underwent metabolic and structural remodeling following differentiation. Optimal HBV infection was achieved in well\u2010differentiated ICOs using spinoculation, with time and donor dependent increase in HBV RNA, cccDNA, extracellular DNA, HBeAg, and HBsAg. Donor dependent drug\u2010responsiveness to entry inhibitor and JAK inhibitor was observed. Despite having a robust ISG response to interferon\u2010\u03b1 and PolyI:C, HBV infection in ICOs did not upregulate ISGs. Human ICOs support HBV infection and replication with donor dependent variation in viral dynamics and cellular responses. 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