OBJECTIVES: Because of the risk of progressive multifocal leukoencephalopathy (PML), natalizumab is generally recommended as second-line treatment for relapsing-remitting multiple sclerosis (RRMS) patients.  For those negative for anti-JC virus antibodies, the natalizumab associated risk of PML is low.  The objective was to estimate the cost-effectiveness of first-line natalizumab versus second-line natalizumab treatment (i.e., initiate glatiramer acetate (GA) then switch to natalizumab) for RRMS patients negative for anti-JC virus antibodies. METHODS: We used a cohort simulation model to estimate the 20-year costs and outcomes of first- and second-line natalizumab treatment.  Model inputs included published natural history progressions of the Expanded Disability Status Scale (EDSS), treatment effects from randomized controlled trials on disease progression and relapse rates, risk of PML, and utilities.  We used the PharMetrics Plus claims database for total costs, switching and discontinuation rates and their associated costs (i.e., first-line treatment with GA then switch or discontinue).  Outputs for the average patient, discounted at 3% per annum, were quality-adjusted life years (QALYs), costs in 2012 US dollars, and incremental cost-effectiveness ratios (ICERs). RESULTS: Compared to natalizumab as second-line treatment after switching from GA, first-line natalizumab treatment was associated with 0.40 incremental QALYs gained, $36,779 more in 20-year costs for an ICER of $91,510 per QALY.  Compared to first-line GA treatment without switching, first-line treatment with natalizumab was associated with an ICER of $95,764 per QALY (likelihood = 0.56 that first-line natalizumab treatment was cost-effective at a willingness-to-pay of $100,000 per QALY). First-line natalizumab treatment dominated second-line natalizumab treatment when compared to GA treatment without switching through principles of extended dominance. CONCLUSIONS: Treating JC virus negative RRMS patients with natalizumab as a first-line treatment provided better value compared to natalizumab use as a second-line agent. More aggressive treatment with natalizumab should be considered for RRMS patients who are negative for anti-JC virus antibodies.