We have previously shown that hypoxic proliferation of human pulmonary microvascular endothelial cells (hPMVEC) depends on epidermal growth factor receptor (EGFR) activation. To determine down-stream signaling leading to proliferation, we tested the hypothesis that hypoxia-induced proliferation in hPMVEC would require EGFR-mediated activation of extracellular signal-regulated kinase (ERK) leading to arginase II induction. To test this hypothesis, hPMVEC were incubated in either normoxia (21% O2, 5% CO2) or hypoxia (1% O2, 5% CO2) and western blotting was performed for EGFR, arginase II, phosphorylated-ERK (pERK), and total ERK (ERK). Hypoxia led to greater EGFR, pERK and arginase II protein levels than did normoxia in hPMVEC. To examine the role of EGFR in these hypoxia-induced changes, hPMVEC were transfected with siRNA against EGFR or a scramble siRNA and placed in hypoxia. Inhibition of EGFR using siRNA attenuated hypoxia-induced pERK and arginase II expression as well as the hypoxia-induced increase in viable cell numbers. hPMVEC were then treated with vehicle, an EGFR inhibitor (AG1478), or an ERK pathway inhibitor (U0126) and placed in hypoxia. Pharmacologic inhibition of EGFR significantly attenuated the hypoxia-induced increase in pERK level. Both AG1478 and U0126 also significantly attenuated the hypoxia-induced increase in viable hPMVEC numbers. hPMVEC were transfected with an adenoviral vector containing arginase II (AdArg2) and overexpression of arginase II rescued the U0126-mediated decrease in viable cell numbers in hypoxic hPMVEC. Our findings suggest that hypoxic activation of EGFR results in phosphorylation of ERK, which is required for hypoxic-induction of arginase II and cellular proliferation.