自然历史博物馆绝不是枯燥乏味的，它的珍藏是惊人的：有眼神狰狞的史前生物、凶猛的古战士，有早已消失的族裔、非洲丛林的动物，还有历史上传奇的英雄人物。这些都被冻结在一个时刻，无论时间怎样流转，它们都不会改变。
赖瑞（本·斯蒂勒饰），一名时运不济的梦想家，他有很多梦想却都无法实现。赖瑞相信总有一天他能成就一番惊人事业。当然，这一切只是梦想，现在摆在眼前的是他必须在圣诞节之前找到工作。否则，他的小儿子就将判由前妻抚养。赖瑞不愿让儿子失望，于是在他的努力之下找到了一份工作——纽约自然历史博物馆的夜间警卫。
工作的第一个晚上，他拿着一个超大手电筒和一本详尽的指示手册，被孤单地留在安静得诡异、庞大的博物馆里。博物馆的老守夜人告诉赖瑞，他的任务就是“确保在晚上没有任何东西能进出博物馆”。赖瑞觉得这个守夜人是老糊涂了，整个博物馆除了他一人之外，还有什么活物呢？等等！那是什么声音？让赖瑞难以置信的是，那些史前生物和只有故事书里才活蹦乱跳的人物，竟一个一个活起来了……
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