Objective The aim of this study is to establish a population pharmacokinetic (PK) model for patients undergoing continuous veno-venous hemodiafiltration (CVVHDF) and optimize the dosing regimen of colistin sulfate. Methods A prospective observational study in a single center was conducted on patients who were administrated with colistin sulfate and CVVHDF for at least 48 h. Blood samples were obtained prior to dosing and four to six blood samples (primarily C 0.5h , C 1h , C 2h , C 4h , and C 6h ) after dosing. The blood concentration of colistin sulfate was determined by ultra-high performance liquid chromatography-tandem mass spectrometry assay. The NONMEM program was used to establish the population PK model and perform Monte Carlo simulations. The predictability and stability of the model were internally evaluated by the goodness of fit plots, visual prediction check, and bootstraps. Results A total of 86 plasma concentrations from 20 patients were used for population PK modeling. A two-compartment model with first-order linear elimination best described the population PK characteristics of colistin sulfate. Cystatin C (CysC) and body weight (WT) were identified as covariates for clearance (CL). Internal evaluation results showed that the final model had good stability and prediction performance. Monte Carlo simulations showed that only when the body WT was 50 kg with CysC ≥3.07 mg/l, and when the body WT was 65 kg with CysC = 5.11 mg/l, and minimum inhibitory concentration (MIC) = 0.25 mg/l, the target attainment probability (PTA) of the daily dose of 1.5 million U regimen was ≥90%. All treatment regimens fail to achieve the target PTA when MIC = 1 mg/l. Conclusions With the decrease of CysC levels and the increase of WT, the dose of colistin sulfate may need to be increased. It may be prudent for colistin sulfate to consider an initial dose doubling and subsequent maintenance dosing regimen of 200–225 million unit daily, administered in 2–3 divided doses, to attain PTA standard. This study was registered at the Chinese Clinical Trial Registry ( www.chictr.org.cn ) (trial registration number ChiCTR2300072191).