Ancient lake deposits in the Mojave Desert indicate that the water cycle in this currently dry place was radically different under past climates. Here we revisit a 700 m core drilled 55 years ago from Searles Valley, California, that recovered evidence for a lacustrine phase during the late Pliocene. We update the paleomagnetic age model and extract new biomarker evidence for climatic conditions from lacustrine deposits (3.373–2.706 Ma). The MBT5Me′ temperature proxy, based on bacterial membrane lipids, detects present-day conditions (21 ± 3 ºC, 1s, n = 2) initially, followed by warmer-than-present conditions (25 ± 3 ºC, n = 17) starting at 3.268 and ending at 2.734 Ma. This is supported by salinity indicators from bacterial and archaeal biomarkers that reveal lake salinity increased after 3.268 Ma. The δ13C values of plant waxes (-30.7 ± 1.4‰, n = 28) are consistent with local C3 taxa, likely expanded conifer woodlands during the pluvial with less C4 than the Pleistocene. dD values (-174 ± 5‰, n = 25) of plant waxes indicate precipitation dD values (‑89 ± 5‰, n = 25) in the late Pliocene are within the same range as the late Pleistocene precipitation dD. Microbial biomarkers identify a deep, freshwater lake and a cooling that corresponds to the onset of major Northern Hemisphere glaciation at marine isotope stage MIS M2. A more saline lake persisted for ~0.6 Ma across the subsequent warmth of the late Pliocene before the lake desiccated at the Pleistocene intensification of Northern Hemisphere Glaciation.