Gabriel Logan and Lian Xing are operatives of The Agency, on the trail of Erich Rhoemer, an international terrorist. In August of 1999, Agent Ellis attempted to infiltrate Rhoemer's narcotics operation in Guácimo, Costa Rica, but was discovered and executed by ex-KGB agent Mara Aramov. Rhoemer then ordered Anton Girdeux, a French mercenary, to burn down the plantation to hide the evidence of their Syphon Filter research.
In Washington, D.C., Edward Benton, the Agency Deputy Director, is briefing Logan's mission to Agency Director Thomas Markinson and a shadowy, unnamed figure. Several weeks later, Gabe and Lian investigate a mysterious viral outbreak in the Himalayas, which leaves only a single survivor. (Lian describes this as impossible.)
The game begins one month after the events in Costa Rica. Rhoemer and his terrorists have launched an assault on Washington, D.C. Gabe defeats Pavel Kravitch, Rhoemer's communications expert, which allows him to gain entry to the Washington Metro. Once inside, an explosive goes off leaving Gabe trapped underground. He pursues Aramov, catching her in the tunnels before heading to a nearby park to aid in the defusing of several viral bombs, where he learns that Aramov has escaped custody. Making his way to the Freedom Memorial, Gabe takes on and defeats Jorge Marcos, Rhoemer's trigger man, before he incinerates Girdeaux.
Lab results show that Syphon Filter is a deadly next-generation biological weapon, capable of targeting specific demographics. Jonathan Phagan, PHARCOM's CEO and founder, attends a gala at his new Expo Center Museum in New York City, where he is taken captive by Aramov and Benton is revealed as a double agent. Gabe kills Benton and frees Phagan, but Aramov, who is captured, again escapes custody.
Gabe goes to Rhoemer's secret base in Rozova, Kazakhstan. He kills Rhoemer's commander Vladislav Gabrek and destroys the base, but Lian is taken captive and presumably killed by Rhoemer.
Gabe is then sent to infiltrate Rhoemer's stronghold in Uzhhorod, Ukraine, which is an abandoned cathedral. Injecting viral patients with what he is convinced is an antidote, Gabe descends deeper into the cathedral to rescue Lian; who explains that there is no 'global' antidote for the virus. They also find Phagan, who is later killed by Aramov. She decides to help Gabe and Lian, since she has nowhere else to go.
Gabe then infiltrates the PHARCOM warehouses in Almaty, Kazakhstan, where PHARCOM security is battling Rhoemer's men. Gabe learns that Markinson, who had been secretly negotiating with Rhoemer to obtain Syphon Filter for the Agency, had made Gabe inject the patients with potassium chloride. Rhoemer reveals he has captured a Soviet-era nuclear ICBM. Rhoemer shoots Markinson and starts the launch sequence for the missile. Gabe cannot prevent it from launching, so his only hope is to initiate its self-destruct sequence once it is in the upper atmosphere. Gabe successfully destroys the missile. Rhoemer, furious at Logan's interference, engages him in a final showdown. Gabe is able to kill him and avert World War III.
In a post-credits scene, the shadowy figure from the beginning of the game is holding a sample of Syphon Filter inside the Agency's headquarters with Aramov, who is laughing.