We have shown that Ca²⁺-mediated protein kinase C (PKC) activation induces impairment of bile salt secretory function and F-actin redistribution in hepatocyte couplets. Because oxidative stress induces Ca²⁺ elevation, we tested here whether PKC inhibition or protein kinase A (PKA) activation, which often counteracts PKC-dependent effects, can prevent and reverse these alterations. The pro-oxidant compounds tert-butylhydroperoxide (tBOOH, 100 μM) and 2,3-dimethoxy-1,4-naphthoquinone (30 μM), reduced by −41% and −29%, respectively, the percentage of couplets accumulating the fluorescent bile salt analog, cholyl-lysylfluorescein in their canalicular vacuoles (p < 0.01). tBOOH-induced bile salt secretory failure was accompanied by internalization of the canalicular bile salt export pump (Bsep), and disarrangement of cytoskeletal F-actin. All these deleterious effects were fully prevented by the intracellular Ca²⁺ chelator BAPTA/AM (20 μM), the pan-specific PKC inhibitors H7 (100 μM) and staurosporine (1 μM), the inhibitor of Ca²⁺-dependent PKCs, Gö6976 (2 μM), and the PKA activator dibutyryl-cAMP (500 μM). H7, Gö6976, and dibutyryl-cAMP not only prevented but also fully reversed the decrease in the cholyl-lysyl-fluorescein accumulation. In conclusion, these results suggest that low levels of oxidative stress impair bile salt secretion by internalizing Bsep through a Ca²⁺-dependent, PKC-mediated mechanism, and that inhibition of PKC, or activation of PKA, prevents and reverses these effects. Alterations in actin organization may be a causal factor.