Mario Molina began his studies at the University of California at Berkeley in 1968, where he would obtain his PhD in physical chemistry. Throughout his years at Berkeley, he participated in various research projects such as the study of molecular dynamics using chemical lasers and investigation of the distribution of internal energy in the products of chemical and photochemical reactions. Throughout this journey is where he worked with his professor and mentor George C. Pimentel who grew his love for chemistry even further. After completing his PhD in physical chemistry, in 1973, he enrolled in a research program at UC Berkeley, with Sherwood Rowland. The topic of interest was Chlorofluorocarbons (CFCs) . The two would later on make one of the largest discoveries in atmospheric chemistry. They developed their theory of ozone depletion, which later influenced the mass public to reduce their use of CFCs. This kickstarted his career as a widely known chemist.
Between 1974 and 2004, Molina variously held research and teaching posts at University of California, Irvine, the Jet Propulsion Laboratory at Caltech, and the Massachusetts Institute of Technology (MIT), where he held a joint appointment in the Department of Earth Atmospheric and Planetary Sciences and the Department of Chemistry. On 1 July 2004, Molina joined the Department of Chemistry and Biochemistry at University of California, San Diego, and the Center for Atmospheric Sciences at the Scripps Institution of Oceanography.
In addition he established a non-profit organization, which opened the Mario Molina Center for Strategic Studies in Energy and the Environment () in Mexico City in 2005. Molina served as its director.
Molina served on the board of trustees for Science Service, now known as Society for Science & the Public, from 2000 to 2005. 
He also served on the board of directors of the John D. and Catherine T. MacArthur Foundation (2004–2014), and as a member of the MacArthur Foundation's Institutional Policy Committee and its Committee on Global Security and Sustainability.
Molina was nominated to the Pontifical Academy of Sciences as of 24 July 2000. He served as a co-chair of the Vatican workshop and co-author of the report Well Under 2 Degrees Celsius: Fast Action Policies to Protect People and the Planet from Extreme Climate Change (2017) with Veerabhadran Ramanathan and Durwood Zaelke.  The report proposed 12 scalable and practical solutions which are part of a three-lever cooling strategy to mitigate climate change.
Molina was named by US president Barack Obama to form a transition team on environmental issues in 2008.
Under President Obama, he was a member of the United States President's Council of Advisors on Science and Technology.
Molina sat on the board of directors for Xyleco.
He contributed to the content of the papal encyclical Laudato Si'.
In 2020, Mario Molina contributed to research regarding the importance of wearing face masks amid the SARS-COV-2 pandemic. The research article titled "Identifying airborne transmission as the dominant route for the spread of COVID-19" was published in the Proceedings of the National Academy of Sciences of the United States of America Journal in collaboration with Renyi Zhang, Yixin Li, Annie L. Zhang and Yuan Wang.