Abstract Vistore® mineral products are hydroxychloride minerals that feature high metal content and improved bioavailability. This study was conducted to compare different sources of copper (Cu) on in vitro rumen fermentation parameters. Three ruminally-cannulated Jersey heifers were adapted to a lactation diet for two weeks before being used as donors. Three sources of Cu at 4 ppm: No supplemental Cu (CON), CuSO4, Vistore Cu, and another Cy hydroxychloride product (Vistore-competitor). The concentration of Cu in this study was selected from a titration study (0 to 8 ppm CuSO4) to identify the minimum concentration of CuSO4 affecting rumen fermentation. The lactation diet (TMR) was dried and ground to 1mm and used as the substrate. Rumen fluid was collected two hours after feeding. Substrate (0.5 g) was inoculated with 100 mL of a 3:1 McDougall’s buffer: ruminal fluid mixture at 39ºC for 24 h. Each treatment was run in triplicate and in three runs. Data were analyzed with R 4.0. The model included fixed effect of treatment and random effect of run. CuSO4 tended to increase lag time (0.78 vs -0.57 h, P = 0.06), reduced (P < 0.05) DMD (52.4 vs. 56.1%), cellulose digestibility (4.9% vs. 41.9%), isobutyrate molar % (0.58 vs. 0.78%) and NH3-N concentration (5.46 vs. 6.91 mg/dL). Vistore and Vistore-competitor maintained the fermentation and digestibility compared to CON. In general, Vistore Cu and Vistore-competitor maintained ruminal fermentation and digestibility parameters while negative effects of CuSO4 were observed. These results indicate different Cu mineral sources may affect the rumen differently.