Objective To investigate the correlation between in vitro release and in vivo absorption of pyridostigmine bromide phospholipid complex nanoemulsion (PPNE) by Loo-Reieglman method. Methods PPNE was prepared and dynamic penetration system was used to analyze its in vitro release. The plasma concentration of pyridostigmine bromide was determined by HPLC after oral administration in rats. Loo-Riegelman method was employed to calculate the in vivo absorption percentage (Fa). Then the regression equation was established between absorption percentage and cumulative release. Results The regression equation was set up as follows: Y=1.376 9X-47.543 2, r=0.9411 (P<0.001). Y represented in vivo absorption percentage and X represented in vitro cumulative release rate. Conclusion There is a significant correlation between in vitro release and in vivo absorption of PPNE.