Ichthyophthirius multifiliis is a ciliated protozoan parasite that infects various freshwater fish， causing ichthyophthiriasis. The life cycle of I. multifiliis comprises three stages： theront， trophont， and tomont. Theront， the only infective stage， invades the host and transforms into the trophont. This study first compared the survival of theronts in different cell culture media to identify the most suitable medium for in vitro culture. Based on these findings， the effects of adding epithelioma papillosum cyprini （EPC） cells and agarose to the culture medium on theront development were investigated using three models： （a）， agarose was positioned in the lower layer beneath the theront and cell mixture； （b）， the agarose mixed directly with theront and cell； and （c），agarose positioned in the upper layer above the theront and cell mixture. The survival rate and size of I. multifiliis were calculated for each condition. The results were as follows： （1） The longest survival time for theronts in medium M199 was up to 6 d. However， theronts were unable to transform into trophonts. （2） The addition of EPC cell aggregates to medium M199 enabled theronts to transformed into trophonts， which survived for 2 d. The size of trophonts was （31.32±3.79） μm on d 2， with no significant growth trend observed. （3） In all three agarose models， theronts was transformed into trophonts and survived for 3 d. The diameters of trophonts in models a， b， and c modes were （37.40±3.99） μm， （39.51±8.51） μm， and （45.14±10.92） μm， respectively， on day 3. In conclusion， the present study demonstrated that adding either EPC cell aggregates or agarose to medium M199 promoted the transformation of theronts into trophonts. However， significant trophonts growth occurred only in the model where the medium was supplemented with both agarose and cells.