Three new hypocrellin derivatives, amino- or amino acid–substituted on the side ring of hypocrellin B (HB), were synthesized by the reactions of HB with 3-methoxypropylamine, 6-aminohexanoic acid and γ-amino-n-butyric acid, respectively. The structures of these compounds were characterized with proton nuclear magnetic resonance spectra, infrared spectra and mass spectra. The UV–visible absorption spectra, singlet oxygen–generating quantum yield and amphiphilicities of hypocrellin derivatives were measured and compared with HB, the parent compound. These derivatives showed strong absorption in the domain of the phototherapeutic window (600–900 nm) and improved amphiphilicity. HB and the derivatives were preliminarily tested for their photodynamic effects on human oral cavity epithelial carcinoma KB cell line in vitro. Two amino acid–substituted hypocrellins showed phototoxicity to the KB cell line. At an inhibitory dosage of 50% killing only 0.51 μmol L⁻¹ compound 3 (or 0.88 μmol L⁻¹ compound 2) and 0.5 J cm⁻² irradiation were required. The hypocrellins exhibited some dark toxicity to the KB cell line. HB and amino acid–substituted hypocrellins showed lower dark toxicity to the KB cell line than amino-substituted hypocrellins in the assessment of cell survival.