Hemolysis in cardiac surgery is often related to the contact of blood with air or artificial surfaces. Variations of negative pressure in the suction cannulas may represent an additional factor. Limited data exist on the contribution of a roller pump‐assisted (RPA) cardiotomy suction unit to hemolysis. Elevation of free hemoglobin (fHb) following air suction (AS) or suction tip occlusion (STO) events of a pump‐assisted cardiotomy suction unit was investigated in a mock circuit filled with blood from slaughtered domestic pigs. AS‐associated hemolysis was measured over 240 minutes with 2 minutes of AS occurring every 10 minutes. STO‐associated hemolysis was analyzed over 80‐minute periods: configuration 1 (c1) comprised a cycle of 20 minutes (min) occlusion and 60 minutes RPA flow (20/60 minutes); c2 comprised 20 cycles of 1/3 minutes; c3 comprised 40 cycles of 0.5/1.5 minutes; and c4 comprised 80 cycles of 0.25/0.75 minutes. The AS setup did not lead to significant hemolysis after 2 (P = .97), 3 (P = .40) or 4 (P = .11) hours. The STO setup showed the greatest hemolysis (ΔfHb of 30 mg/dL) in c1 after 20 minutes. ΔfHb was different in c1 from all other configurations at 20 minutes (P < .0001) and 80 minutes (P < .05). Ex vivo generation of large negative pressures by STO events is the main cause of cardiotomy suction‐associated hemolysis. The clinical relevance of this mechanism needs further investigations.