Aim To investigate the role of selective inhibition of the PI3K/Akt/mTOR signaling pathway on prompting rabbit primary macrophage autophagy. Methods Primary macrophages were obtained intraperitoneally from the New Zealand rabbits and then were co-cultured with the phosphatidylinositol 3-kinase （PI3K） inhibitor LY294002 （10 μmol/L）, protein kinase B （Akt） inhibitor triciribine （20 μmol/L）, mammalian target of rapamycin （mTOR） inhibitor rapamycin （10 nmol/L） and no drugs respectively for 12 hours. Ultrastructural changes of macrophages were examined by transmission electron microscopy. The expression level of microtubule-associated protein light chain 3Ⅱ（LC3Ⅱ） was assayed by immunofluorescence. Protein expression levels of Akt, mTOR, phosphorylation of protein kinase B （p-Akt）， phosphorylation of mammalian target of rapamycin （p-mTOR） and autophage related protein Beclin-1 and autophagy protein 5 and 12 conjugated form （Atg5-Atg12） were measured by Western blot. Mondansylcadaverine （MDC） staining was used to see autophagy lysosome changes. Results Compared with the control group, few autophagosomes and vacuoles were detected in group LY294002 while plenty of typical autophagosomes were detected in group rapamcin and triciribine. The expression levels of Beclin-1 and Atg5-Atg12 decreased significantly in group rapamcin, while increased significantly in group rapamcin and triciribine. The fluorescence microscope showed few dots of LC3Ⅱ in group LY294002 and many in group rapamcin and triciribine. The expression of p-Akt and p-mTOR increased obviously in group rapamcin while the former increased a lot and the latter decreased in group rapacin after co-culturing of 4 hours. The expression of p-mTOR decreased significantly in the treated groups, however, p-Akt decreased significantly in group rapamcin and triciribine but increased obviously in group rapamci after 12 hours’ co-culture. There were no significant differences on the total AKT and mTOR levels among the treated groups. MDC staining showed decreased autophagic lysosomes in group LY294002 and increased autophagic lysosomes in group rapamcin and triciribine. Conclusion Selective inhibition of PI3K/Akt/mTOR signaling pathway can promote rabbit primary macrophage autophagy while inhibition of PI3K suppress macrophage autophagy by other signaling pathway.