Curculigo orchioides Gaertn is a herbaceous plant that has long been used as a tonic in Vietnam with noticeable health benefits. However, the demand for rhizomes of this species could not be met due to their decreasing number in natural habitats. Despite its vulnerability, there are still not enough researches on producing calli of C. orchioides, which is a method having the capacity of creating a large source of cell biomass for bioactive compounds’ extractions. Thus, this study was conducted to figure out the best conditions for C. orchioides’s callus proliferation. Different light regimes, mineral media, and concentrations of some factors like kinetin (KIN), α-naphthaleneacetic acid (NAA), yeast extract (YE), activated charcoal (AC), and silver nitrate (AgNO3) were examined. It is shown by the results that half-strength MS medium (½ MS) given 0.5 mg/L KIN and MS medium supplemented with 0.5 mg/L KIN and put in the dark (0 light hour : 24 dark hours) are the optimal conditions for callus proliferation, with the highest fresh weights (FWs), dry weights (DWs), and growth indices (GIs) of 3.89 g / 0.45 g / 7.78, and 4.10 g / 0.47 g / 8.20, respectively. Additionally, the inhibitory effects of AgNO3, YE, and AC were demonstrated since there was no observed heavy callus in the media containing those factors.