Introduction Administration of retinal gene- and stem cell therapy in patients with retinal degenerative diseases (RDD) is in many cases dependent of a subretinal approach. It has been indicated that manual subretinal injection is associated with outer retinal damage, which may be explained be high flow rate in the injection cannula. In the present porcine study, we evaluated flow-related retinal damage after controlled subretinal injection at different flow rates. Methods Flow rate through a 41G cannula was estimated at different injection pressures (6-48 PSI (pounds per square inch)) in an in-vitro setup. A linear correlation between flow rate and injection pressure was found from 6-32 PSI. In full anesthesia, 12 pigs were vitrectomized and received a controlled subretinal injection of 300 microliters balanced saline solution at injection pressures of either 14, 24 and 32 PSI (four in each group). Prior to surgery and two and four weeks after surgery, the eyes where examined by multifocal electroretinogram (mfERG) and fundus photographs. At the end of follow-up, the eyes were enucleated for histology. Results The in vitro flow study determined that the flow in a 41 G cannula shift from laminar to tubular at 32 PSI, and that the manual injection flow is tubular. In the porcine study we showed a significant difference in retinal pigment epithelium (RPE) damage between the three pressure groups (p = 0.0096). There was no significant difference in damage to the outer retina (p = 0.1526), but the high-pressure group (32 PSI) had most outer retinal damage. The middle-pressure group (24 PSI) showed minimum retinal damage. There was no significant change in the mfERG ratios during follow-up. Discussion/Conclusion This study indicates that an injection pressure at approximately 24 PSI might be safe for subretinal delievery. Retinal damage at low injection pressures may be explained by mechanical damage to the RPE due to prolonged needle time in the subretinal space, whilst retinal damage at high pressures can be related to high flow in the injection cannula. Controlled subretinal injection pressure of 24 PSI showed minimum mechanical and flow-related damage to the porcine retina.