Objective To determine the effects of macrophages on the survival and axonal regeneration of retinal ganglion cells (RGC) in vitro. Methods A rat co -culture model was established with RGC and peritoneal cavity macrophages in transwell chambers. RGC cultured without macrophages served as the control. The axonal growth and survival time of RGC in the model were observed under phase contrast microscopy. The survival of RGC was determined by counting the cells with Trypan blue staining. The average lengths of the processes of RGC cultured on days 1, 3 and 5 were measured and calculated. Results The average counts of viable cells with Trypan blue staining in the co-culture system were 35.50±2.92, 28.20±3.36, 18.70±3.95, and 8.80±1.55 on days 1, 3, 5 and 7, respectively. And there were no statistically significant differences when compared to the controls (36.20±2.35, 27.10±2.96, 15.80±3.04, and 8.40±2.01, respectively;t=0.369, 0.497, 0.487, and 2.854; P>0.05). The average lengths of the RGC processes in the co-culture system were (19.79±3.98)μm, (68.30±4.07)μn, and (95.51 ±6.5l)μm on days 1, 3 and 5, respectively, which were significantly longer when compared to the controls [(15.28±1.28)μm, (58.18±4.22)μm, and (82.61 ±3.75)μm, respectively; t =-4.562, -6.554, -7.027; P<0.05). Conclusion Results shows that macrophages could significantly promote the axonal regeneration of RGC in a co-culture system, but there is no obvious effect on cell survival.