Microfiltration (MF) can effectively remove microorganisms and somatic cells from milk, increasing the safety and shelf life of milk and dairy products. The main challenge in MF is membrane fouling, which leads to a significant decline in permeate flux over time. This work aimed to elucidate the mechanisms of membrane fouling in cold (6C) MF of skim milk and optimize a CO2 backpulsing technique that can diminish membrane fouling by physically removing the foulant from the membrane surface. Using 3 injection ports, a CO2 injection frequency of 120 s and an injection duration of 1 s, a permeate flux of 30.02 ± 0.48 L/m2h was obtained after 3 h of MF at a cross-flow velocity of 3.8 m/s, as compared to 25.85 ± 0.99 L/m2h for the control. Additionally, a smaller drop in flux was observed for the optimized CO2 backpulsing process (15.57% after 3 h) as compared to the control (29.83% after 3 h). When MF was conducted at 6 m/s, the permeate flux after 3 h was 82.32 ± 4.42 L/m2h for MF with CO2 backpulsing, as compared to 71.39 ± 4.21 L/m2h for the control. MF with CO2 backpulsing also resulted in a higher transmission of total solids and protein than the control MF. A systematic investigation of the mechanisms of membrane fouling was conducted by identifying the proteins and minerals in four foulant streams: weakly attached external foulants (We), weakly attached internal foulants (Wi), strongly attached external foulants (Se), and strongly attached internal foulants (Si). The foulants were evaluated both after MF (without and with CO2 backpulsing) and after a brief contact between the membrane and milk (adsorption study). The concentration of minerals was very small in all foulant streams, below 2.5 ppm, and likely they do not contribute significantly to membrane fouling in cold MF. Proteomics analysis showed that all major milk proteins were present in all foulant streams. In adsorption trial, [alpha]lactalbumin level in We was higher than in milk, which indicates an affinity of this protein for the membrane material. The serum proteins [alpha]-lactalbumin and bovine serum albumin (BSA) were found in a higher proportion in the "weakly attached" fractions (We and Wi) from the instantaneous adsorption study as compared to the control MF (without CO2 backpulsing), which suggests that caseins were mainly introduced into the fouling layer when transmembrane pressure was applied. Casein concentration did not increase in the foulants from CO2 backpulsing MF, despite the localized decrease in pH caused by the contact with CO 2. More significantly, CO2 backpulsing reduced the total protein concentration in We, with 52.98 ± 4.87 [mu]g/mL for CO2 backpulsing MF as compared to 62.20 ± 10.13 [mu]g/mL for control MF. Overall, the data indicates that CO2 backpulsing resulted in less weakly attached external foulants and possibly less loosely deposited materials on the membrane surface as compared to control MF, which improved permeation through the membrane. The knowledge generated in this study could be used to identify solutions to further minimize membrane fouling and increase the efficiency of milk MF. The CO2 backpulsing technique could be an efficient and affordable solution to mitigate fouling, and is also applicable to other cold membrane filtration processes, such as the MF of juice or beer.