Layer-by-layer (LbL) deposited polyelectrolyte thin films containing insulin were prepared and the pH-triggered release of insulin was studied. Insulin-containing LbL films were successfully prepared by the alternate deposition of insulin and poly(vinyl sulfate) (PVS), poly(acrylic acid) (PAA), or dextran sulfate (DS) in acidic solutions (pH 1.0–3.0) through the electrostatic forces of attraction between positively-charged insulin and polyanions. The loading of insulin in the film was dependent on the type of polyanions used and the highest insulin loading was observed when PAA was used as the polyanion. Insulin-containing LbL films were decomposed when the films were exposed to weakly acidic (pH 5.0–6.0) or neutral solutions (pH 7.4) due to a loss of electrostatic forces of attraction between the insulin and polyanions in the films, which in turn was caused by the charge reversal of insulin from positive to negative in the weakly acidic or neutral media. Thus, insulin is released from LbL films at a neutral or weakly acidic pH. Circular dichroism (CD) spectroscopy revealed that the released insulin retains its original secondary structure. The insulin-containing LbL films were found to be satisfactorily stable even in the presence of a digestive enzyme (pepsin) at pH 1.4 (stomach pH). Consequently, the potential use of the insulin-containing LbL films is suggested for future application in the oral delivery of insulin.